Reduction of Astrogliosis by Early Treatment of Pneumococcal Meningitis Measured by Simultaneous Imaging, In Vivo, of the Pathogen and Host
http://www.100md.com
感染与免疫杂志 2005年第12期
Xenogen Corporation, 860 Atlantic Avenue, Alameda, California 94501
ABSTRACT
We developed a method for simultaneous in vivo biophotonic monitoring of pneumococcal meningitis and the accompanying neuronal injury in live transgenic mice. Streptococcus pneumoniae engineered for bioluminescence (lux) was used for direct visualization of disease progression and antibiotic treatment in a mouse model of meningitis. The host response was monitored in transgenic mice containing an inducible firefly luciferase (luc) reporter gene under transcriptional control of the mouse glial fibrillary acidic protein (GFAP) promoter. Based on the different spectra of light emission and substrate requirements for lux and luc, we were able to separately monitor the two reporters using a highly sensitive in vivo imaging system. The level of neuronal damage and recovery following antibiotic treatment was dependent on the time of treatment. This model has potential for simultaneous multiparameter monitoring and testing of therapies that target the pathogen or host response to prevent neuronal injury and recovery.
INTRODUCTION
Bacterial meningitis is a severe inflammatory disease of the central nervous system (CNS) that leads to long-term neurological sequelae or death in many patients, even when it is successfully treated with antibiotics (12, 21, 28). About 30% of the survivors of pneumococcal meningitis, the most frequent form of bacterial meningitis in adults, develop long-term cognitive impairments, including sensory motor deficits, cerebral palsy, seizure disorders, mental retardation, and learning impairments (12, 21, 28, 34). Data from animal models of meningitis and postmortem studies of human brains indicate that both necrotic and apoptotic neuronal injury are responsible for many of the neurologic deficits seen after bacterial meningitis (34, 35).
Several animal models of bacterial meningitis have recently been used to identify the bacterial and host inflammatory factors responsible for brain injury during the course of bacterial meningitis (7, 10, 19, 28, 37), but the development of feasible therapeutic strategies to decrease meningitis-induced brain dysfunction remains a challenge (12). One major limitation with animal models has been the difficulty in assessing the molecular mechanisms of both the inflammatory response and the neuronal damage over time without sacrificing the animal. Even a simple evaluation of tissue damage in these models usually requires time-consuming histological examination of selected postmortem tissue samples rather than a whole-body analysis.
We combined animal engineering with modern imaging technology to visualize the multiparameter dynamics of specific molecular processes in living animals. Using recent advances in biophotonic imaging of bioluminescent reporters for detection for the study of human disease in live animal models (1, 4) and the introduction of reporter genes into genetically modified animals (15, 16), we demonstrated that it is possible to investigate both the disease process and the host response through the activity of a given gene in the context of a living organism.
Astrocytes have many roles in the brain and are the predominant neuroglial cells of the CNS (18, 20). They are integral parts of synapses and provide physical support to neighboring neurons, meninges, and vasculature. Glial fibrillary acidic protein (GFAP) is a major intermediate structural filament protein that is expressed predominantly in mature astrocytes of the CNS and is considered to be a reliable cell-specific biomarker for monitoring neuronal activity under development and pathological conditions (3, 17, 18, 24, 30, 31, 36). While the molecular mechanism of astrocyte activation is poorly understood, a biomarker for astrogliosis is the cytoskeletal GFAP of astrocytes (24). Consequently, understanding the in vivo activity of GFAP during pneumococcal meningitis is of interest for obtaining insight into astrocyte function and for understanding the CNS response to infection. To enable visualization and quantification of GFAP activity in response to infection in a single animal without surgery or biopsy, we used a transgenic mouse model that involves the firefly luciferase gene (luc) driven by the murine GFAP promoter (36). Using this GFAP animal model, we produced meningitis by infecting animals with a lux-containing bacterium for simultaneous in vivo analysis of disease progression and the GFAP response. Here we describe a novel model and approach that allowed noninvasive tracking of an infection and the host response to the disease agent and therapy, which integrated the concept of longitudinal analysis of spatial and temporal data acquired from individual animals nondestructively. While previously this approach was not possible with standard models, we believe that it can be used to answer very diverse questions about biological events and the host response, including questions about other types of pathogenesis, tumors, and degenerative diseases.
MATERIALS AND METHODS
Bacterial strain and growth conditions. The bacterial strain used in this study was Streptococcus pneumoniae A 66.1 (type 3) that was made bioluminescent by integration of a modified lux operon into its chromosome (5); the resulting strain was designated S. pneumoniae Xen 10. Integration of the lux cassette into the bacterial chromosome had no effect on the bacterial pathogenicity or survival in mice. The organism was grown in brain heart infusion supplemented with 10% fetal calf serum, and the inoculum was prepared as previously described (10).
Mouse model of meningitis. A total of 29 female FVB/N-Tg (GFAP-luc) mice (36) (Xenogen Corp, Alameda, CA) weighing 22 to 30 g were anesthetized with 2.5% isoflurane, and their heads and spine areas were shaved to expose the inoculation sites, which allowed accurate delivery of the pathogen to the subarachnoid space. Using a 30.5-gauge needle and a Hamilton syringe, 10 μl of a suspension containing 104 CFU of S. pneumoniae Xen 10 in sterile pyrogen-free saline was slowly delivered intracisternally as described previously (9). Another group of mice was mock infected with sterile saline, and these mice served as uninfected controls. Mice were observed until they recovered from anesthesia, at which time they showed no evidence of behavioral abnormalities. At different times after infection (11 h, 17 h, and 19 h), groups of mice (n = 5) were treated subcutaneously with 100 mg/kg ceftriaxone (7, 9) in 0.1 ml saline. The treatment was repeated every 12 h for 3 days (a total of six treatments). The MIC and minimal bactericidal concentration of ceftriaxone for S. pneumoniae Xen 10 were 0.015 and 0.03 μg/ml, respectively (7, 9). A limited number of mice from each experimental group were euthanized, and cerebrospinal fluid (CSF) was withdrawn at several times after infection as described previously (9) for determination of the bacterial titers in the CSF. All experimental procedures for the animals were performed in accordance with guidelines of the Institutional Animal Care and Use Committee. Moribund mice were sacrificed according to the Institutional Animal Care and Use Committee protocol, and the time of sacrifice was considered to be the time of death.
GFAP-luc transgenic mice. The GFAP-luc transgenic mouse line was created by microinjection of a luciferase reporter driven by a 12-kb fragment of the GFAP promoter into FVB/N embryos at Xenogen Biosciences Corporation, as described previously (36).
Spectral measurements using the IVIS 200 imaging system. In vivo bioluminescence imaging was performed using an IVIS imaging system (200 series; Xenogen Corp., Alameda, CA). The IVIS 200 imaging system used in this study is equipped with 17 band-pass filters that are 20 nm wide, with central wavelengths ranging from 420 nm to 740 nm. Since the bioluminescence of bacterial luciferase (lux) and the bioluminescence of firefly luciferase (luc) are separated spectrally (4), different filters were used to selectively image the bioluminescent signals from bacteria (lux) and GFAP promoter-driven (luc) reporter expression (host response). The results of these experiments showed that bacterial and host spectra were separated spectrally, with peaks at 490 and 610 nm, respectively (Fig. 1). Prior to imaging, mice were anesthetized with 2 to 2.5% isoflurane gas and then placed in an imaging box without restraint and imaged for a maximum of 5 min at various times. During imaging, mice were placed on the warmed stage of the light-tight imaging chamber and maintained in an anesthetized state by constant delivery of 2.5% isoflurane through an IVIS anesthesia manifold. Images were acquired using the 500-nm filter to target the bacterial luciferase (max, 490 nm) and 620-nm filters to measure firefly luciferase (max, 610 nm). In addition, images were taken before and after intraperitoneal injection of 150 mg/kg luciferin (BioSynth, Staad, Switzerland). The images acquired before luciferin injection collected only light emitted from pneumococci since these organisms did not require an external supply of luciferin to produce light (4). After luciferin injection, images recorded with the 500-nm filter revealed only bacterial luciferase since firefly luciferase emission was negligible in this region of the spectrum. Following luciferin injection, images collected at 620 nm were a combination of the host luciferase expression and the bacterial luciferase expression. Since bacterial luciferase has a broad spectrum, a small fraction of its light was still collected with the 620-nm filter. In order to separate host expression in the 620-nm-filtered images, the bacterial contribution was subtracted using the mathematical tools available in the Living Image 2.5 software (Xenogen Corp.). First, the total flux was measured for both the 500-nm and 620-nm images acquired before luciferin injection in regions of interest around the brain or spine, and the ratio of the 620-nm bacterial luciferase emission to the 500-nm bacterial luciferase emission was determined. This ratio was used as a weighing coefficient to subtract the 500-nm images from the 620-nm images acquired after luciferin injection. This subtraction allowed quantification of the host bioluminescence and the bacterial bioluminescence individually. The light output from specified regions of interest was quantified by determining the total number of photons emitted per second using the Living Image analysis software. The data were represented as pseudo-color images indicating light intensity (blue or black, least intense; red or yellow, most intense), which were superimposed over the grayscale reference photographs. The IVIS imaging system was calibrated with a NIST traceable source in order to measure light in absolute, quantitative physical units.
Histologic procedures. Postharvest brains or spines from infected or control animals were fixed in 4% formalin in phosphate-buffered saline (pH 7.0) and shipped to Pathology Associates, CRL (Frederick, MD) for analysis. Paraffin-embedded tissues were cut to obtain approximately 5-μm sections, which were processed for staining with hematoxylin and eosin, Gram stain, or immunohistochemical GFAP. Histopathologic examination and interpretation of data were performed by a veterinary pathologist.
Statistical analyses. Statistical analyses were carried out by using Student's t test. A P value of 0.05 was considered significant.
RESULTS
Simultaneous monitoring of bacterial and host responses. Concurrent monitoring of the host response and bacterial burden in the CNS of a single animal is possible because the spectra for lux and luc are different, with peaks at 490 and 610 nm, respectively (Fig. 1). Second, the insect luciferase produces light only when its substrate, luciferin, is provided, whereas bioluminescently engineered bacteria emit light continuously without an exogenous substrate (4, 5). These spectral and substrate differences between lux and luc allowed both reporters to be used in the same animal to monitor two different events, bacterial growth and GFAP activity. Based on luciferin kinetic profiles, we concluded that luc measurements could be made 12 min after luciferin injection and that imaging could continue for up to 45 min at each wavelength after a single injection of the substrate. These studies also demonstrated that the bioavailability of luciferin in the brain was similar in healthy and inflamed blood-brain barriers, confirming its suitability for in vivo use, as shown previously (25). Real-time in vivo biophotonic images of bacterial and GFAP signals from representative animals from the control group and the groups of animals treated with antibiotic are shown in Fig. 2.
Real-time visualization of infection and response to antibiotic therapy. The total photon emission from the brain and spine following intracisternal inoculation of mice with S. pneumoniae was quantified using the Living Image software, and the cumulative results are shown in Fig. 3A and B. The bacterial signal intensity first increased exponentially in the brain and then expanded in a time-dependent manner down the spinal cord (Fig. 2), indicating that there was growth and spread of bacteria beyond the site of inoculation, as shown previously (9). The photon intensity in the brain and spine reached the peak levels around 24 h postinfection (Fig. 3A and B). With the increase in signal intensity, animals became severely lethargic, opisthotonos, and moribund, indicating that there was an association between disease severity and photon intensity. Consistent with the bioluminescent signal data, the bacterial titer in the CSF also increased with time; at 11 h the titer was 7.5 x 103 CFU/μl, at 17 h it was 1.2 x 105 CFU/μl, and at 19 h it was 2 x 105 CFU/μl. Compared to saline-treated mice, a rapid decline in the bioluminescent signal was observed in the ceftriaxome-treated groups at 11 or 17 h after infection. After two treatments the signal was almost undetectable, suggesting that the infection was eradicated (Fig. 2 and 3). A ceftriaxone-induced reduction in bioluminescence in animals treated after 19 h was also noticed. However, the decline appeared to be slower than the declines seen in groups that were treated 11 h and 17 h after infection (Fig. 3A and B). The colony counts for infected CSF paralleled the intensities of bacterial bioluminescence measured in both treated and untreated animals, as shown in our previous studies (9), demonstrating that bioluminescence is a convenient method for monitoring an infection and the response to treatment (8, 11). There was no bacterial growth from CSF that did not have a detectable bioluminescent signal following antibiotic therapy for any of the treated groups. This indicated the effectiveness of ceftriaxone for rapid sterilization of CSF, as shown previously (9). All mice treated early in infection (i.e., at 11 h and 17 h) survived without any clinical signs of disease, whereas 80% of the mice died within 48 h despite treatment when therapy was delayed until 19 h into the infection, indicating that the mortality is dependent on the time between infection and the initiation of treatment. No animals that were not treated survived beyond 30 h. Control animals that were inoculated with sterile saline showed no bioluminescent signal or altered health status.
Real-time visualization of GFAP and response to antibiotic therapy. Figure 4A and B show the total photon emission for GFAP from brains and spines from the group of mice with meningitis shown in Fig. 3A and B. Approximately 3 to 4 h after the appearance of a bacterial signal, a host signal was first noticed in the head region, and it progressed parallel to the bacterial signal, suggesting that the neuronal damage was not limited to the brain but extended down the spinal column (Fig. 2 and 4A and B). Compared to noninfected control mice, the photon intensities for regions of interest in the head and spine increased exponentially over the next few hours and reached approximately 108 photons/s around the brain by an average of 40-fold (P 0.05) and 107 photons/s by an average of 12-fold (P 0. 02) around the spine after 24 h (Fig. 4A and B). The rapid increase in the GFAP signal 3 to 4 h following infection could be prevented by starting antibiotic treatment of the infected animals by 11 h postinfection. If the same treatment was postponed until 17 h postinfection, when the GFAP signal reached moderately high levels in the brain (3.8 x 107 photons/s), the bacterial signal was rapidly reduced (Fig. 3A and B). However, this treatment regimen failed to reduce the GFAP signal in the brain to the same level as the 11-h postinfection treatment regimen (Fig. 4A). The mice treated at 17 h postinfection showed no further increase in GFAP signal, but the signal persisted for the next 30 h compared to untreated mice and declined gradually by the end of the study. Even with the subsequent decline, the GFAP signal remained approximately fourfold higher than the signal for the uninfected group at the end of study (Fig. 4A). The GFAP photon intensity for the spinal region of this group after administration of the antibiotic started to decline and slowly reverted almost to the intensity observed for the noninfected control group by the end of the study (Fig. 4B). Remarkably, administration of the antibiotic to animals with severe meningitis, when the bacterial load, disease severity, and GFAP signal were high (19 h postinfection), resulted in a robust increase in the intensity of GFAP, to levels even above the levels for the untreated control group. In contrast to the bacterial signal, despite effective bacterial sterilization, the intensity of the GFAP signal continued to increase and reached approximately 30- and 100-fold in the brain and spine at 22 and 41 h, respectively (Fig. 2 and 4). In this group, only 20% of the animals survived beyond 22 h. The signal intensities in the brain and spinal column in the surviving animals declined but remained greater than the intensities in the noninfected control mice. The mice that survived pneumococcal infection in this group experienced infrequent epileptic seizures. No bacteria were recovered from the blood or CSF of the dead or live animals in groups given ceftriaxone, which suggests that death was not due to the presence of bacteria.
To better understand whether the photons were emitted from the CNS, the brains and spinal columns were removed immediately following in vivo imaging and imaged ex vivo to determine both bacterial and GFAP signals. Figure 5 shows the brain of an untreated mouse after 19 h of meningitis. These images show that the majority of the bacterial signal was restricted to regions in the brain where CSF was trapped within ventricular spaces (dorsal) or to the site of injection (ventral). In contrast, the GFAP signal appeared to spread over the entire brain, with brighter regions in specific parts in the ventral or dorsal region, indicating that differential expression of GFAP occurred in these regions. Up-regulation of GFAP in the brains from postmeningitic animals was further confirmed by histology.
Histopathology. Brains and spinal tissue from mice that were given saline (control) or mice that were infected with S. pneumoniae and then left untreated or treated with antibiotic were removed at various times after infection and analyzed by histopathology. The immunohistochemically stained sections shown in Fig. 6A to C are sections from an infected untreated mouse with severe meningitis after 24 h. Numerous chains of gram-positive cocci were present in areas of necrosis, and there were multiple foci within the meninges bordering areas of necrosis (Fig. 6A). Several areas of liquifactive necrosis admixed with microabscesses were present in the brain stem beneath the cerebellum (Fig. 6B). Compared to noninfected animals (Fig. 6D), the GFAP activity in brain sections from an infected animal was multifocally increased in regions bordering the areas of necrosis (Fig. 6C). Thus, the data obtained by biophotonic imaging were consistent with the presence of neuronal damage determined histologically, which supported the hypothesis that GFAP activity in transgenic mice accurately reflects astocyte activity and serves as a convenient in situ marker for GFAP.
DISCUSSION
In this study we demonstrated for the first time that it is possible to monitor bioluminescence from an infecting pathogen and the host response to the infectious process concurrently in the same living animal. We accomplished this by producing a model for meningitis in a genetically modified mouse model expressing an astrocyte-specific biomarker (36). We then developed a method for simultaneous in vivo biophotonic monitoring of pneumococcal meningitis and accompanying expression of GFAP in activated astrocytes as a surrogate marker for neuronal injury in live mice. This dual monitoring allowed us to track the progression of infection at the same time that we tracked the induction of the host response through the use of two different bioluminescent reporter genes and a low-light imaging system. The novelty of our work lies not only in visualization of the progression of infection in real time in intact animals but also in the ability to detect and monitor the infection-associated neuronal damage as it occurs in a living animal throughout the disease. One bioluminescent reporter was specific for S. pneumoniae carrying a complete lux operon, and the second bioluminescent reporter was in a transgenic mouse line carrying firefly luciferase under transcriptional control of the GFAP promoter. By using spectral characteristics and substrate requirements for the two reporter genes, we were able to concurrently visualize the GFAP signal as an indicator of damage to the CNS during progressive replication and the spread of bacteria within the brains and spinal cords of diseased animals. This approach allowed us to determine the temporal, sequential, and spatial distribution of the pathogen and the host response to infection in real time. In addition, we used the model to assess the effect of ceftriaxone, the standard therapeutic agent for pneumococcal meningitis, and the timing of treatment on the outcome of disease, as well as on the expression of the GFAP. Treatment of infected mice at different times after infection (11 h, 17 h, and 19 h) led to a rapid decrease in the bacterial burden, resulting in quick sterilization and curing of the infection. However, as the bacterial load decreased, a massive immediate increase in the GFAP signal intensity occurred in direct relation to the bacterial load. The greatest increase in the GFAP signal in the brain and spinal column occurred in groups that were treated 19 h after infection was initiated, and this increase did not occur in groups treated earlier (11 h and 17 h) or in animals that received no antibiotic. The lysis and killing of pneumococci by the -lactam antibiotic in the first few hours of treatment may have contributed to the accompanying transient increase in expression of GFAP that appeared to be more pronounced with a higher concentration of pneumococci in the subarachnoid space. Previous findings indeed demonstrated that treatment of bacterial meningitis with -lactam antibiotics liberates harmful bacterial products (peptidoglycans, teichoic and lipoteichoic acids, pneumolysin) in the subarachnoid space, which results in an immediate increase in the number of active molecules that are capable of causing a rapid increase in the inflammatory response (7, 10, 21, 28, 32). In fact, the release of bacterial components, as well as host proinflammatory components and mediators, has been shown to be neurotoxic, causing apoptosis in neurons, predominantly in the dentate gyrus of the hippocampus (13, 21, 37). Interestingly, treatment with antibiotics such as rifampin and clindamycin, which induce death without lysis by inhibiting bacterial protein synthesis, reduced the mortality and neuronal injury in mice with pneumococcal meningitis compared with treatment with the -lactam antibiotic ceftriaxone (2, 22). The immediate massive up-modulation of GFAP following treatment of mice with a high bacterial burden demonstrated that the response of astrocytes to neurologic injury is very rapid. The factors that initiate injury-induced astrocyte activation appeared to act within 1 h of focal injury. Interestingly, ceftriaxone has been shown to be neuroprotective in a nonbacterial disease, amyotrophic lateral sclerosis (26). The protective mechanism of the action of the antibiotic in this neurodegenerative disease is believed to be mediated through increased activation of the promoter for the gene for the neurotransmitter glutamate. The pathway for the promoter activation is unclear.
Reduction of the GFAP signal to a level resembling that of the uninfected control groups was seen in mice treated early, when they had a low bacterial load and clinical signs, suggesting that the damage to the CNS was minimal and the astrocytes were capable of faster recovery. In contrast, the most prominent GFAP signals were the signals from groups of animals that had advanced meningitis (and a heavy bacterial load) but were treated and cured with a bactericidal agent. In these animals not only was GFAP expressed for a longer time, but the levels remained elevated above the control group levels throughout the observation period. Most strikingly, the animal that survived beyond 42 h in this group also experienced occasional seizures. Imaging of dissected brains ex vivo revealed that the GFAP signal came from specific parts of the brain with various degrees of intensity. The degree of resolution could be critical when the neurobiology of pneumococcal infection is studied, as it allows correlations to be made between particular symptoms and the focus of the infection in the brain. For instance, pneumococcus-associated neuronal injury most frequently occurs in the hippocampus, an area of the brain associated with spatial verbal memory, as well as learning (7, 27, 29, 33, 37).
In addition to the structural function of GFAP, this protein has also been implicated in several processes in the CNS, including maintenance of the blood-brain barrier, neuroprotection, volume regulation, myelination, neuromodulation, and the ability to protect the CNS from infection (3, 14, 30, 31). Moreover, it plays a role in restoring structural and physiological integrity after injury (23). Unlike other neurons in the brain, hippocampal dentate gyrus neurons have the ability to undergo proliferation and neurogenesis (24). Our observation of a persistent, elevated GFAP signal in postmeningitic mice suggests that GFAP may be involved in recovery from injury.
In previous studies injury to the CNS in bacterial meningitis has been determined primarily by histology, immunohistochemistry, or magnetic resonance imaging volumetry (6, 7, 19, 35, 37). In most instances, the data are obtained at terminal sampling points or at autopsy, which does not allow the disease course to be monitored in the same animal and comparative values to be assessed. Unfortunately, these approaches do not allow investigation of the connection between the host and the pathogen during the acute phase of infection or during convalescence. In this regard, our approach provides the intriguing possibility of being able to visualizing the two processes simultaneously and quantitatively in real time in the same animal by whole-body imaging. It effectively monitors astrocyte activity and facilitates the analysis of early astrogliosis. Understanding the pathogenic mechanisms that lead to neuronal injury that accompany pneumococcal meningitis is crucial for the development of more effective therapeutic strategies. This work provides such information on the molecular targets for possible pharmacological intervention that could be used to manipulate systems for improvement of neuroprotection and repair in a damaged CNS.
ACKNOWLEDGMENTS
We thank C. Dalesio (Graphics and Communications, Xenogen Corporation) for assistance with drawings.
REFERENCES
1. Bhaumik, S., X. Z. Lewis, and S. S. Gambhir. 2004. Optical imaging of Renilla luciferase, synthetic Renilla luciferase, and firefly luciferase reporter gene expression in living mice. J. Biomed. Opt. 9:578-586.
2. Bottcher, T., H. Ren, M. Goiny, J. Gerber, J. Lykkesfeldt, U. Kuhnt, M. Lotz, S. Bunkowski, C. Werner, I. Schau, A. Spreer, S. Christen, and R. Nau. 2004. Clindamycin is neuroprotective in experimental Streptococcus pneumoniae meningitis compared with ceftriaxone. J. Neurochem. 91:1450-1460.
3. Brenner, M., and A. Messing. 1996. GFAP transgenic mice. Methods 10:351-364.
4. Contag, C. H., and M. H. Bachmann. 2002. Advances in in vivo bioluminescence imaging of gene expression. Annu. Rev. Biomed Eng. 4:235-260.
5. Francis, K. P., J. Yu, C. Bellinger-Kawahara, D. Joh, M. J. Hawkinson, G. Xiao, T. F. Purchio, M. G. Caparon, M. Lipsitch, and P. R. Contag. 2001. Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon. Infect. Immun. 69:3350-3358.
6. Free, S. L., L. M. Li, D. R. Fish, S. D. Shorvon, and J. M. Stevens. 1996. Bilateral hippocampal volume loss in patients with a history of encephalitis or meningitis. Epilepsia 37:400-405.
7. Gerber, J., G. Raivich, A. Wellmer, C. Noeske, T. Kunst, A. Werner, W. Bruck, and R. Nau. 2001. A mouse model of Streptococcus pneumoniae meningitis mimicking several features of human disease. Acta Neuropathol. 101:499-508.
8. Kadurugamuwa, J. L., K. Modi, Yu, J., K. P. Francis, T. Purchio, and P. R. Contag. 2005. Noninvasive biophotonic imaging for monitoring of catheter-associated urinary tract infections and therapy in mice. Infect. Immun. 73:3878-3887.
9. Kadurugamuwa, J. L., K. Modi, Yu, J., K. P. Francis, C. Orihuela, E. Tuomanen, A. F. Purchio, and P. R. Contag. 2005. Noninvasive monitoring of pneumococcal meningitis and evaluation of treatment efficacy in an experimental mouse model. Mol. Imaging 4:137-142.
10. Kadurugamuwa, J. L., B. Hengstler, and O. Zak. 1989. Cerebrospinal fluid protein profile in experimental pneumococcal meningitis and its alteration by ampicillin and anti-inflammatory agents. J. Infect. Dis. 159:26-34.
11. Kadurugamuwa, J. L., L. V. Sin, J. Yu, K. P. Francis, R. Kimura, T. Purchio, and P. R. Contag. 2003. Rapid direct method for monitoring antibiotics in a mouse model of bacterial biofilm infection. Antimicrob. Agents Chemother. 47:3130-3137.
12. Kim, K. S. 2003. Pathogenesis of bacterial meningitis: from bacteraemia to neuronal injury. Nat. Rev. Neurosci. 4:376-385.
13. Kim, Y. S., S. Kennedy, and M. G. Tauber. 1995. Toxicity of Streptococcus pneumoniae in neurons, astrocytes, and microglia in vitro. J. Infect. Dis. 171:1363-1368.
14. Lejon, V., L. E. Rosengren, P. Buscher, J. E. Karlsson, and H. N. Sema. 1999. Detection of light subunit neurofilament and glial fibrillary acidic protein in cerebrospinal fluid of Trypanosoma brucei gambiense-infected patients. Am. J. Trop. Med. Hyg. 60:94-98.
15. Maggi, A., L. Ottobrini, A. Biserni, G. Lucignani, and P. Ciana. 2004. Techniques: reporter mice—a new way to look at drug action. Trends Pharmacol. Sci. 25:337-342.
16. Massoud, T. F., and S. S. Gambhir. 2003. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev. 17:545-580.
17. Messing, A., M. W. Head, K. Galles, E. J. Galbreath, J. E. Goldman, and M. Brenner. 1998. Fatal encephalopathy with astrocyte inclusions in GFAP transgenic mice. Am. J. Pathol. 152:391-398.
18. Miller, G. 2005. Neuroscience. The dark side of glia. Science 308:778-781.
19. Mitchell, L., S. H. Smith, J. S. Braun, K. H. Herzog, J. R. Weber, and E. I. Tuomanen. 2004. Dual phases of apoptosis in pneumococcal meningitis. J. Infect. Dis. 190:2039-2046.
20. Mucke, L., M. B. Oldstone, J. C. Morris, and M. I. Nerenberg. 1991. Rapid activation of astrocyte-specific expression of GFAP-lacZ transgene by focal injury. New Biol. 3:465-474.
21. Nau, R., and W. Bruck. 2002. Neuronal injury in bacterial meningitis: mechanisms and implications for therapy. Trends Neurosci. 25:38-45.
22. Nau, R., A. Wellmer, A. Soto, K. Koch, O. Schneider, H. Schmidt, J. Gerber, U. Michel, and W. Bruck. 1999. Rifampin reduces early mortality in experimental Streptococcus pneumoniae meningitis. J. Infect. Dis. 179:1557-1560.
23. Pekny, M., and M. Pekna. 2004. Astrocyte intermediate filaments in CNS pathologies and regeneration. J. Pathol. 204:428-437.
24. Raivich, G., M. Bohatschek, C. U. Kloss, A. Werner, L. L. Jones, and G. W. Kreutzberg. 1999. Neuroglial activation repertoire in the injured brain: graded response, molecular mechanisms and cues to physiological function. Brain Res. Brain Res. Rev. 30:77-105.
25. Rehemtulla, A., D. E. Hall, L. D. Stegman, U. Prasad, G. Chen, M. S. Bhojani, T. L. Chenevert, and B. D. Ross. 2002. Molecular imaging of gene expression and efficacy following adenoviral-mediated brain tumor gene therapy. Mol. Imaging 1:43-55.
26. Rothstein, J. D., S. Patel, M. R. Regan, C. Haenggeli, Y. H. Huang, D. E. Bergles, L. Jin, M. Dykes Hoberg, S. Vidensky, D. S. Chung, S. V. Toan, L. I. Bruijn, Z. Z. Su, P. Gupta, and P. B. Fisher. 2005. Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature 433:73-77.
27. Sass, K. J., D. D. Spencer, J. H. Kim, M. Westerveld, R. A. Novelly, and T. Lencz. 1990. Verbal memory impairment correlates with hippocampal pyramidal cell density. Neurology 40:1694-1697.
28. Scheld, W. M., U. Koedel, B. Nathan, and H. W. Pfister. 2002. Pathophysiology of bacterial meningitis: mechanism(s) of neuronal injury. J. Infect. Dis. 186(Suppl. 2):S225-S233.
29. Sherry, D. F., L. F. Jacobs, and S. J. Gaulin. 1992. Spatial memory and adaptive specialization of the hippocampus. Trends Neurosci. 15:298-303.
30. Sousa Vde, O., L. Romao, V. M. Neto, and F. C. Gomes. 2004. Glial fibrillary acidic protein gene promoter is differently modulated by transforming growth factor-beta 1 in astrocytes from distinct brain regions. Eur. J. Neurosci. 19:1721-1730.
31. Stenzel, W., S. Soltek, D. Schluter, and M. Deckert. 2004. The intermediate filament GFAP is important for the control of experimental murine Staphylococcus aureus-induced brain abscess and Toxoplasma encephalitis. J. Neuropathol. Exp. Neurol. 63:631-640.
32. Tuomanen, E., B. Hengstler, R. Rich, M. A. Bray, O. Zak, and A. Tomasz. 1987. Nonsteroidal anti-inflammatory agents in the therapy for experimental pneumococcal meningitis. J. Infect. Dis. 155:985-990.
33. Vaher, P. R., V. N. Luine, E. Gould, and B. S. McEwen. 1994. Effects of adrenalectomy on spatial memory performance and dentate gyrus morphology. Brain Res. 656:71-78.
34. van der Flier, M., S. P. Geelen, J. L. Kimpen, I. M. Hoepelman, and E. I. Tuomanen. 2003. Reprogramming the host response in bacterial meningitis: how best to improve outcome Clin. Microbiol. Rev. 16:415-429.
35. Wellmer, A., C. Noeske, J. Gerber, U. Munzel, and R. Nau. 2000. Spatial memory and learning deficits after experimental pneumococcal meningitis in mice. Neurosci. Lett. 296:137-140.
36. Zhu, L., S. Ramboz, D. Hewitt, L. Boring, D. S. Grass, and A. F. Purchio. 2004. Non-invasive imaging of GFAP expression after neuronal damage in mice. Neurosci. Lett. 367:210-212.
37. Zysk, G., W. Bruck, J. Gerber, Y. Bruck, H. W. Prange, and R. Nau. 1996. Anti-inflammatory treatment influences neuronal apoptotic cell death in the dentate gyrus in experimental pneumococcal meningitis. J. Neuropathol. Exp. Neurol. 55:722-728.(Jagath L. Kadurugamuwa, K)
ABSTRACT
We developed a method for simultaneous in vivo biophotonic monitoring of pneumococcal meningitis and the accompanying neuronal injury in live transgenic mice. Streptococcus pneumoniae engineered for bioluminescence (lux) was used for direct visualization of disease progression and antibiotic treatment in a mouse model of meningitis. The host response was monitored in transgenic mice containing an inducible firefly luciferase (luc) reporter gene under transcriptional control of the mouse glial fibrillary acidic protein (GFAP) promoter. Based on the different spectra of light emission and substrate requirements for lux and luc, we were able to separately monitor the two reporters using a highly sensitive in vivo imaging system. The level of neuronal damage and recovery following antibiotic treatment was dependent on the time of treatment. This model has potential for simultaneous multiparameter monitoring and testing of therapies that target the pathogen or host response to prevent neuronal injury and recovery.
INTRODUCTION
Bacterial meningitis is a severe inflammatory disease of the central nervous system (CNS) that leads to long-term neurological sequelae or death in many patients, even when it is successfully treated with antibiotics (12, 21, 28). About 30% of the survivors of pneumococcal meningitis, the most frequent form of bacterial meningitis in adults, develop long-term cognitive impairments, including sensory motor deficits, cerebral palsy, seizure disorders, mental retardation, and learning impairments (12, 21, 28, 34). Data from animal models of meningitis and postmortem studies of human brains indicate that both necrotic and apoptotic neuronal injury are responsible for many of the neurologic deficits seen after bacterial meningitis (34, 35).
Several animal models of bacterial meningitis have recently been used to identify the bacterial and host inflammatory factors responsible for brain injury during the course of bacterial meningitis (7, 10, 19, 28, 37), but the development of feasible therapeutic strategies to decrease meningitis-induced brain dysfunction remains a challenge (12). One major limitation with animal models has been the difficulty in assessing the molecular mechanisms of both the inflammatory response and the neuronal damage over time without sacrificing the animal. Even a simple evaluation of tissue damage in these models usually requires time-consuming histological examination of selected postmortem tissue samples rather than a whole-body analysis.
We combined animal engineering with modern imaging technology to visualize the multiparameter dynamics of specific molecular processes in living animals. Using recent advances in biophotonic imaging of bioluminescent reporters for detection for the study of human disease in live animal models (1, 4) and the introduction of reporter genes into genetically modified animals (15, 16), we demonstrated that it is possible to investigate both the disease process and the host response through the activity of a given gene in the context of a living organism.
Astrocytes have many roles in the brain and are the predominant neuroglial cells of the CNS (18, 20). They are integral parts of synapses and provide physical support to neighboring neurons, meninges, and vasculature. Glial fibrillary acidic protein (GFAP) is a major intermediate structural filament protein that is expressed predominantly in mature astrocytes of the CNS and is considered to be a reliable cell-specific biomarker for monitoring neuronal activity under development and pathological conditions (3, 17, 18, 24, 30, 31, 36). While the molecular mechanism of astrocyte activation is poorly understood, a biomarker for astrogliosis is the cytoskeletal GFAP of astrocytes (24). Consequently, understanding the in vivo activity of GFAP during pneumococcal meningitis is of interest for obtaining insight into astrocyte function and for understanding the CNS response to infection. To enable visualization and quantification of GFAP activity in response to infection in a single animal without surgery or biopsy, we used a transgenic mouse model that involves the firefly luciferase gene (luc) driven by the murine GFAP promoter (36). Using this GFAP animal model, we produced meningitis by infecting animals with a lux-containing bacterium for simultaneous in vivo analysis of disease progression and the GFAP response. Here we describe a novel model and approach that allowed noninvasive tracking of an infection and the host response to the disease agent and therapy, which integrated the concept of longitudinal analysis of spatial and temporal data acquired from individual animals nondestructively. While previously this approach was not possible with standard models, we believe that it can be used to answer very diverse questions about biological events and the host response, including questions about other types of pathogenesis, tumors, and degenerative diseases.
MATERIALS AND METHODS
Bacterial strain and growth conditions. The bacterial strain used in this study was Streptococcus pneumoniae A 66.1 (type 3) that was made bioluminescent by integration of a modified lux operon into its chromosome (5); the resulting strain was designated S. pneumoniae Xen 10. Integration of the lux cassette into the bacterial chromosome had no effect on the bacterial pathogenicity or survival in mice. The organism was grown in brain heart infusion supplemented with 10% fetal calf serum, and the inoculum was prepared as previously described (10).
Mouse model of meningitis. A total of 29 female FVB/N-Tg (GFAP-luc) mice (36) (Xenogen Corp, Alameda, CA) weighing 22 to 30 g were anesthetized with 2.5% isoflurane, and their heads and spine areas were shaved to expose the inoculation sites, which allowed accurate delivery of the pathogen to the subarachnoid space. Using a 30.5-gauge needle and a Hamilton syringe, 10 μl of a suspension containing 104 CFU of S. pneumoniae Xen 10 in sterile pyrogen-free saline was slowly delivered intracisternally as described previously (9). Another group of mice was mock infected with sterile saline, and these mice served as uninfected controls. Mice were observed until they recovered from anesthesia, at which time they showed no evidence of behavioral abnormalities. At different times after infection (11 h, 17 h, and 19 h), groups of mice (n = 5) were treated subcutaneously with 100 mg/kg ceftriaxone (7, 9) in 0.1 ml saline. The treatment was repeated every 12 h for 3 days (a total of six treatments). The MIC and minimal bactericidal concentration of ceftriaxone for S. pneumoniae Xen 10 were 0.015 and 0.03 μg/ml, respectively (7, 9). A limited number of mice from each experimental group were euthanized, and cerebrospinal fluid (CSF) was withdrawn at several times after infection as described previously (9) for determination of the bacterial titers in the CSF. All experimental procedures for the animals were performed in accordance with guidelines of the Institutional Animal Care and Use Committee. Moribund mice were sacrificed according to the Institutional Animal Care and Use Committee protocol, and the time of sacrifice was considered to be the time of death.
GFAP-luc transgenic mice. The GFAP-luc transgenic mouse line was created by microinjection of a luciferase reporter driven by a 12-kb fragment of the GFAP promoter into FVB/N embryos at Xenogen Biosciences Corporation, as described previously (36).
Spectral measurements using the IVIS 200 imaging system. In vivo bioluminescence imaging was performed using an IVIS imaging system (200 series; Xenogen Corp., Alameda, CA). The IVIS 200 imaging system used in this study is equipped with 17 band-pass filters that are 20 nm wide, with central wavelengths ranging from 420 nm to 740 nm. Since the bioluminescence of bacterial luciferase (lux) and the bioluminescence of firefly luciferase (luc) are separated spectrally (4), different filters were used to selectively image the bioluminescent signals from bacteria (lux) and GFAP promoter-driven (luc) reporter expression (host response). The results of these experiments showed that bacterial and host spectra were separated spectrally, with peaks at 490 and 610 nm, respectively (Fig. 1). Prior to imaging, mice were anesthetized with 2 to 2.5% isoflurane gas and then placed in an imaging box without restraint and imaged for a maximum of 5 min at various times. During imaging, mice were placed on the warmed stage of the light-tight imaging chamber and maintained in an anesthetized state by constant delivery of 2.5% isoflurane through an IVIS anesthesia manifold. Images were acquired using the 500-nm filter to target the bacterial luciferase (max, 490 nm) and 620-nm filters to measure firefly luciferase (max, 610 nm). In addition, images were taken before and after intraperitoneal injection of 150 mg/kg luciferin (BioSynth, Staad, Switzerland). The images acquired before luciferin injection collected only light emitted from pneumococci since these organisms did not require an external supply of luciferin to produce light (4). After luciferin injection, images recorded with the 500-nm filter revealed only bacterial luciferase since firefly luciferase emission was negligible in this region of the spectrum. Following luciferin injection, images collected at 620 nm were a combination of the host luciferase expression and the bacterial luciferase expression. Since bacterial luciferase has a broad spectrum, a small fraction of its light was still collected with the 620-nm filter. In order to separate host expression in the 620-nm-filtered images, the bacterial contribution was subtracted using the mathematical tools available in the Living Image 2.5 software (Xenogen Corp.). First, the total flux was measured for both the 500-nm and 620-nm images acquired before luciferin injection in regions of interest around the brain or spine, and the ratio of the 620-nm bacterial luciferase emission to the 500-nm bacterial luciferase emission was determined. This ratio was used as a weighing coefficient to subtract the 500-nm images from the 620-nm images acquired after luciferin injection. This subtraction allowed quantification of the host bioluminescence and the bacterial bioluminescence individually. The light output from specified regions of interest was quantified by determining the total number of photons emitted per second using the Living Image analysis software. The data were represented as pseudo-color images indicating light intensity (blue or black, least intense; red or yellow, most intense), which were superimposed over the grayscale reference photographs. The IVIS imaging system was calibrated with a NIST traceable source in order to measure light in absolute, quantitative physical units.
Histologic procedures. Postharvest brains or spines from infected or control animals were fixed in 4% formalin in phosphate-buffered saline (pH 7.0) and shipped to Pathology Associates, CRL (Frederick, MD) for analysis. Paraffin-embedded tissues were cut to obtain approximately 5-μm sections, which were processed for staining with hematoxylin and eosin, Gram stain, or immunohistochemical GFAP. Histopathologic examination and interpretation of data were performed by a veterinary pathologist.
Statistical analyses. Statistical analyses were carried out by using Student's t test. A P value of 0.05 was considered significant.
RESULTS
Simultaneous monitoring of bacterial and host responses. Concurrent monitoring of the host response and bacterial burden in the CNS of a single animal is possible because the spectra for lux and luc are different, with peaks at 490 and 610 nm, respectively (Fig. 1). Second, the insect luciferase produces light only when its substrate, luciferin, is provided, whereas bioluminescently engineered bacteria emit light continuously without an exogenous substrate (4, 5). These spectral and substrate differences between lux and luc allowed both reporters to be used in the same animal to monitor two different events, bacterial growth and GFAP activity. Based on luciferin kinetic profiles, we concluded that luc measurements could be made 12 min after luciferin injection and that imaging could continue for up to 45 min at each wavelength after a single injection of the substrate. These studies also demonstrated that the bioavailability of luciferin in the brain was similar in healthy and inflamed blood-brain barriers, confirming its suitability for in vivo use, as shown previously (25). Real-time in vivo biophotonic images of bacterial and GFAP signals from representative animals from the control group and the groups of animals treated with antibiotic are shown in Fig. 2.
Real-time visualization of infection and response to antibiotic therapy. The total photon emission from the brain and spine following intracisternal inoculation of mice with S. pneumoniae was quantified using the Living Image software, and the cumulative results are shown in Fig. 3A and B. The bacterial signal intensity first increased exponentially in the brain and then expanded in a time-dependent manner down the spinal cord (Fig. 2), indicating that there was growth and spread of bacteria beyond the site of inoculation, as shown previously (9). The photon intensity in the brain and spine reached the peak levels around 24 h postinfection (Fig. 3A and B). With the increase in signal intensity, animals became severely lethargic, opisthotonos, and moribund, indicating that there was an association between disease severity and photon intensity. Consistent with the bioluminescent signal data, the bacterial titer in the CSF also increased with time; at 11 h the titer was 7.5 x 103 CFU/μl, at 17 h it was 1.2 x 105 CFU/μl, and at 19 h it was 2 x 105 CFU/μl. Compared to saline-treated mice, a rapid decline in the bioluminescent signal was observed in the ceftriaxome-treated groups at 11 or 17 h after infection. After two treatments the signal was almost undetectable, suggesting that the infection was eradicated (Fig. 2 and 3). A ceftriaxone-induced reduction in bioluminescence in animals treated after 19 h was also noticed. However, the decline appeared to be slower than the declines seen in groups that were treated 11 h and 17 h after infection (Fig. 3A and B). The colony counts for infected CSF paralleled the intensities of bacterial bioluminescence measured in both treated and untreated animals, as shown in our previous studies (9), demonstrating that bioluminescence is a convenient method for monitoring an infection and the response to treatment (8, 11). There was no bacterial growth from CSF that did not have a detectable bioluminescent signal following antibiotic therapy for any of the treated groups. This indicated the effectiveness of ceftriaxone for rapid sterilization of CSF, as shown previously (9). All mice treated early in infection (i.e., at 11 h and 17 h) survived without any clinical signs of disease, whereas 80% of the mice died within 48 h despite treatment when therapy was delayed until 19 h into the infection, indicating that the mortality is dependent on the time between infection and the initiation of treatment. No animals that were not treated survived beyond 30 h. Control animals that were inoculated with sterile saline showed no bioluminescent signal or altered health status.
Real-time visualization of GFAP and response to antibiotic therapy. Figure 4A and B show the total photon emission for GFAP from brains and spines from the group of mice with meningitis shown in Fig. 3A and B. Approximately 3 to 4 h after the appearance of a bacterial signal, a host signal was first noticed in the head region, and it progressed parallel to the bacterial signal, suggesting that the neuronal damage was not limited to the brain but extended down the spinal column (Fig. 2 and 4A and B). Compared to noninfected control mice, the photon intensities for regions of interest in the head and spine increased exponentially over the next few hours and reached approximately 108 photons/s around the brain by an average of 40-fold (P 0.05) and 107 photons/s by an average of 12-fold (P 0. 02) around the spine after 24 h (Fig. 4A and B). The rapid increase in the GFAP signal 3 to 4 h following infection could be prevented by starting antibiotic treatment of the infected animals by 11 h postinfection. If the same treatment was postponed until 17 h postinfection, when the GFAP signal reached moderately high levels in the brain (3.8 x 107 photons/s), the bacterial signal was rapidly reduced (Fig. 3A and B). However, this treatment regimen failed to reduce the GFAP signal in the brain to the same level as the 11-h postinfection treatment regimen (Fig. 4A). The mice treated at 17 h postinfection showed no further increase in GFAP signal, but the signal persisted for the next 30 h compared to untreated mice and declined gradually by the end of the study. Even with the subsequent decline, the GFAP signal remained approximately fourfold higher than the signal for the uninfected group at the end of study (Fig. 4A). The GFAP photon intensity for the spinal region of this group after administration of the antibiotic started to decline and slowly reverted almost to the intensity observed for the noninfected control group by the end of the study (Fig. 4B). Remarkably, administration of the antibiotic to animals with severe meningitis, when the bacterial load, disease severity, and GFAP signal were high (19 h postinfection), resulted in a robust increase in the intensity of GFAP, to levels even above the levels for the untreated control group. In contrast to the bacterial signal, despite effective bacterial sterilization, the intensity of the GFAP signal continued to increase and reached approximately 30- and 100-fold in the brain and spine at 22 and 41 h, respectively (Fig. 2 and 4). In this group, only 20% of the animals survived beyond 22 h. The signal intensities in the brain and spinal column in the surviving animals declined but remained greater than the intensities in the noninfected control mice. The mice that survived pneumococcal infection in this group experienced infrequent epileptic seizures. No bacteria were recovered from the blood or CSF of the dead or live animals in groups given ceftriaxone, which suggests that death was not due to the presence of bacteria.
To better understand whether the photons were emitted from the CNS, the brains and spinal columns were removed immediately following in vivo imaging and imaged ex vivo to determine both bacterial and GFAP signals. Figure 5 shows the brain of an untreated mouse after 19 h of meningitis. These images show that the majority of the bacterial signal was restricted to regions in the brain where CSF was trapped within ventricular spaces (dorsal) or to the site of injection (ventral). In contrast, the GFAP signal appeared to spread over the entire brain, with brighter regions in specific parts in the ventral or dorsal region, indicating that differential expression of GFAP occurred in these regions. Up-regulation of GFAP in the brains from postmeningitic animals was further confirmed by histology.
Histopathology. Brains and spinal tissue from mice that were given saline (control) or mice that were infected with S. pneumoniae and then left untreated or treated with antibiotic were removed at various times after infection and analyzed by histopathology. The immunohistochemically stained sections shown in Fig. 6A to C are sections from an infected untreated mouse with severe meningitis after 24 h. Numerous chains of gram-positive cocci were present in areas of necrosis, and there were multiple foci within the meninges bordering areas of necrosis (Fig. 6A). Several areas of liquifactive necrosis admixed with microabscesses were present in the brain stem beneath the cerebellum (Fig. 6B). Compared to noninfected animals (Fig. 6D), the GFAP activity in brain sections from an infected animal was multifocally increased in regions bordering the areas of necrosis (Fig. 6C). Thus, the data obtained by biophotonic imaging were consistent with the presence of neuronal damage determined histologically, which supported the hypothesis that GFAP activity in transgenic mice accurately reflects astocyte activity and serves as a convenient in situ marker for GFAP.
DISCUSSION
In this study we demonstrated for the first time that it is possible to monitor bioluminescence from an infecting pathogen and the host response to the infectious process concurrently in the same living animal. We accomplished this by producing a model for meningitis in a genetically modified mouse model expressing an astrocyte-specific biomarker (36). We then developed a method for simultaneous in vivo biophotonic monitoring of pneumococcal meningitis and accompanying expression of GFAP in activated astrocytes as a surrogate marker for neuronal injury in live mice. This dual monitoring allowed us to track the progression of infection at the same time that we tracked the induction of the host response through the use of two different bioluminescent reporter genes and a low-light imaging system. The novelty of our work lies not only in visualization of the progression of infection in real time in intact animals but also in the ability to detect and monitor the infection-associated neuronal damage as it occurs in a living animal throughout the disease. One bioluminescent reporter was specific for S. pneumoniae carrying a complete lux operon, and the second bioluminescent reporter was in a transgenic mouse line carrying firefly luciferase under transcriptional control of the GFAP promoter. By using spectral characteristics and substrate requirements for the two reporter genes, we were able to concurrently visualize the GFAP signal as an indicator of damage to the CNS during progressive replication and the spread of bacteria within the brains and spinal cords of diseased animals. This approach allowed us to determine the temporal, sequential, and spatial distribution of the pathogen and the host response to infection in real time. In addition, we used the model to assess the effect of ceftriaxone, the standard therapeutic agent for pneumococcal meningitis, and the timing of treatment on the outcome of disease, as well as on the expression of the GFAP. Treatment of infected mice at different times after infection (11 h, 17 h, and 19 h) led to a rapid decrease in the bacterial burden, resulting in quick sterilization and curing of the infection. However, as the bacterial load decreased, a massive immediate increase in the GFAP signal intensity occurred in direct relation to the bacterial load. The greatest increase in the GFAP signal in the brain and spinal column occurred in groups that were treated 19 h after infection was initiated, and this increase did not occur in groups treated earlier (11 h and 17 h) or in animals that received no antibiotic. The lysis and killing of pneumococci by the -lactam antibiotic in the first few hours of treatment may have contributed to the accompanying transient increase in expression of GFAP that appeared to be more pronounced with a higher concentration of pneumococci in the subarachnoid space. Previous findings indeed demonstrated that treatment of bacterial meningitis with -lactam antibiotics liberates harmful bacterial products (peptidoglycans, teichoic and lipoteichoic acids, pneumolysin) in the subarachnoid space, which results in an immediate increase in the number of active molecules that are capable of causing a rapid increase in the inflammatory response (7, 10, 21, 28, 32). In fact, the release of bacterial components, as well as host proinflammatory components and mediators, has been shown to be neurotoxic, causing apoptosis in neurons, predominantly in the dentate gyrus of the hippocampus (13, 21, 37). Interestingly, treatment with antibiotics such as rifampin and clindamycin, which induce death without lysis by inhibiting bacterial protein synthesis, reduced the mortality and neuronal injury in mice with pneumococcal meningitis compared with treatment with the -lactam antibiotic ceftriaxone (2, 22). The immediate massive up-modulation of GFAP following treatment of mice with a high bacterial burden demonstrated that the response of astrocytes to neurologic injury is very rapid. The factors that initiate injury-induced astrocyte activation appeared to act within 1 h of focal injury. Interestingly, ceftriaxone has been shown to be neuroprotective in a nonbacterial disease, amyotrophic lateral sclerosis (26). The protective mechanism of the action of the antibiotic in this neurodegenerative disease is believed to be mediated through increased activation of the promoter for the gene for the neurotransmitter glutamate. The pathway for the promoter activation is unclear.
Reduction of the GFAP signal to a level resembling that of the uninfected control groups was seen in mice treated early, when they had a low bacterial load and clinical signs, suggesting that the damage to the CNS was minimal and the astrocytes were capable of faster recovery. In contrast, the most prominent GFAP signals were the signals from groups of animals that had advanced meningitis (and a heavy bacterial load) but were treated and cured with a bactericidal agent. In these animals not only was GFAP expressed for a longer time, but the levels remained elevated above the control group levels throughout the observation period. Most strikingly, the animal that survived beyond 42 h in this group also experienced occasional seizures. Imaging of dissected brains ex vivo revealed that the GFAP signal came from specific parts of the brain with various degrees of intensity. The degree of resolution could be critical when the neurobiology of pneumococcal infection is studied, as it allows correlations to be made between particular symptoms and the focus of the infection in the brain. For instance, pneumococcus-associated neuronal injury most frequently occurs in the hippocampus, an area of the brain associated with spatial verbal memory, as well as learning (7, 27, 29, 33, 37).
In addition to the structural function of GFAP, this protein has also been implicated in several processes in the CNS, including maintenance of the blood-brain barrier, neuroprotection, volume regulation, myelination, neuromodulation, and the ability to protect the CNS from infection (3, 14, 30, 31). Moreover, it plays a role in restoring structural and physiological integrity after injury (23). Unlike other neurons in the brain, hippocampal dentate gyrus neurons have the ability to undergo proliferation and neurogenesis (24). Our observation of a persistent, elevated GFAP signal in postmeningitic mice suggests that GFAP may be involved in recovery from injury.
In previous studies injury to the CNS in bacterial meningitis has been determined primarily by histology, immunohistochemistry, or magnetic resonance imaging volumetry (6, 7, 19, 35, 37). In most instances, the data are obtained at terminal sampling points or at autopsy, which does not allow the disease course to be monitored in the same animal and comparative values to be assessed. Unfortunately, these approaches do not allow investigation of the connection between the host and the pathogen during the acute phase of infection or during convalescence. In this regard, our approach provides the intriguing possibility of being able to visualizing the two processes simultaneously and quantitatively in real time in the same animal by whole-body imaging. It effectively monitors astrocyte activity and facilitates the analysis of early astrogliosis. Understanding the pathogenic mechanisms that lead to neuronal injury that accompany pneumococcal meningitis is crucial for the development of more effective therapeutic strategies. This work provides such information on the molecular targets for possible pharmacological intervention that could be used to manipulate systems for improvement of neuroprotection and repair in a damaged CNS.
ACKNOWLEDGMENTS
We thank C. Dalesio (Graphics and Communications, Xenogen Corporation) for assistance with drawings.
REFERENCES
1. Bhaumik, S., X. Z. Lewis, and S. S. Gambhir. 2004. Optical imaging of Renilla luciferase, synthetic Renilla luciferase, and firefly luciferase reporter gene expression in living mice. J. Biomed. Opt. 9:578-586.
2. Bottcher, T., H. Ren, M. Goiny, J. Gerber, J. Lykkesfeldt, U. Kuhnt, M. Lotz, S. Bunkowski, C. Werner, I. Schau, A. Spreer, S. Christen, and R. Nau. 2004. Clindamycin is neuroprotective in experimental Streptococcus pneumoniae meningitis compared with ceftriaxone. J. Neurochem. 91:1450-1460.
3. Brenner, M., and A. Messing. 1996. GFAP transgenic mice. Methods 10:351-364.
4. Contag, C. H., and M. H. Bachmann. 2002. Advances in in vivo bioluminescence imaging of gene expression. Annu. Rev. Biomed Eng. 4:235-260.
5. Francis, K. P., J. Yu, C. Bellinger-Kawahara, D. Joh, M. J. Hawkinson, G. Xiao, T. F. Purchio, M. G. Caparon, M. Lipsitch, and P. R. Contag. 2001. Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon. Infect. Immun. 69:3350-3358.
6. Free, S. L., L. M. Li, D. R. Fish, S. D. Shorvon, and J. M. Stevens. 1996. Bilateral hippocampal volume loss in patients with a history of encephalitis or meningitis. Epilepsia 37:400-405.
7. Gerber, J., G. Raivich, A. Wellmer, C. Noeske, T. Kunst, A. Werner, W. Bruck, and R. Nau. 2001. A mouse model of Streptococcus pneumoniae meningitis mimicking several features of human disease. Acta Neuropathol. 101:499-508.
8. Kadurugamuwa, J. L., K. Modi, Yu, J., K. P. Francis, T. Purchio, and P. R. Contag. 2005. Noninvasive biophotonic imaging for monitoring of catheter-associated urinary tract infections and therapy in mice. Infect. Immun. 73:3878-3887.
9. Kadurugamuwa, J. L., K. Modi, Yu, J., K. P. Francis, C. Orihuela, E. Tuomanen, A. F. Purchio, and P. R. Contag. 2005. Noninvasive monitoring of pneumococcal meningitis and evaluation of treatment efficacy in an experimental mouse model. Mol. Imaging 4:137-142.
10. Kadurugamuwa, J. L., B. Hengstler, and O. Zak. 1989. Cerebrospinal fluid protein profile in experimental pneumococcal meningitis and its alteration by ampicillin and anti-inflammatory agents. J. Infect. Dis. 159:26-34.
11. Kadurugamuwa, J. L., L. V. Sin, J. Yu, K. P. Francis, R. Kimura, T. Purchio, and P. R. Contag. 2003. Rapid direct method for monitoring antibiotics in a mouse model of bacterial biofilm infection. Antimicrob. Agents Chemother. 47:3130-3137.
12. Kim, K. S. 2003. Pathogenesis of bacterial meningitis: from bacteraemia to neuronal injury. Nat. Rev. Neurosci. 4:376-385.
13. Kim, Y. S., S. Kennedy, and M. G. Tauber. 1995. Toxicity of Streptococcus pneumoniae in neurons, astrocytes, and microglia in vitro. J. Infect. Dis. 171:1363-1368.
14. Lejon, V., L. E. Rosengren, P. Buscher, J. E. Karlsson, and H. N. Sema. 1999. Detection of light subunit neurofilament and glial fibrillary acidic protein in cerebrospinal fluid of Trypanosoma brucei gambiense-infected patients. Am. J. Trop. Med. Hyg. 60:94-98.
15. Maggi, A., L. Ottobrini, A. Biserni, G. Lucignani, and P. Ciana. 2004. Techniques: reporter mice—a new way to look at drug action. Trends Pharmacol. Sci. 25:337-342.
16. Massoud, T. F., and S. S. Gambhir. 2003. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev. 17:545-580.
17. Messing, A., M. W. Head, K. Galles, E. J. Galbreath, J. E. Goldman, and M. Brenner. 1998. Fatal encephalopathy with astrocyte inclusions in GFAP transgenic mice. Am. J. Pathol. 152:391-398.
18. Miller, G. 2005. Neuroscience. The dark side of glia. Science 308:778-781.
19. Mitchell, L., S. H. Smith, J. S. Braun, K. H. Herzog, J. R. Weber, and E. I. Tuomanen. 2004. Dual phases of apoptosis in pneumococcal meningitis. J. Infect. Dis. 190:2039-2046.
20. Mucke, L., M. B. Oldstone, J. C. Morris, and M. I. Nerenberg. 1991. Rapid activation of astrocyte-specific expression of GFAP-lacZ transgene by focal injury. New Biol. 3:465-474.
21. Nau, R., and W. Bruck. 2002. Neuronal injury in bacterial meningitis: mechanisms and implications for therapy. Trends Neurosci. 25:38-45.
22. Nau, R., A. Wellmer, A. Soto, K. Koch, O. Schneider, H. Schmidt, J. Gerber, U. Michel, and W. Bruck. 1999. Rifampin reduces early mortality in experimental Streptococcus pneumoniae meningitis. J. Infect. Dis. 179:1557-1560.
23. Pekny, M., and M. Pekna. 2004. Astrocyte intermediate filaments in CNS pathologies and regeneration. J. Pathol. 204:428-437.
24. Raivich, G., M. Bohatschek, C. U. Kloss, A. Werner, L. L. Jones, and G. W. Kreutzberg. 1999. Neuroglial activation repertoire in the injured brain: graded response, molecular mechanisms and cues to physiological function. Brain Res. Brain Res. Rev. 30:77-105.
25. Rehemtulla, A., D. E. Hall, L. D. Stegman, U. Prasad, G. Chen, M. S. Bhojani, T. L. Chenevert, and B. D. Ross. 2002. Molecular imaging of gene expression and efficacy following adenoviral-mediated brain tumor gene therapy. Mol. Imaging 1:43-55.
26. Rothstein, J. D., S. Patel, M. R. Regan, C. Haenggeli, Y. H. Huang, D. E. Bergles, L. Jin, M. Dykes Hoberg, S. Vidensky, D. S. Chung, S. V. Toan, L. I. Bruijn, Z. Z. Su, P. Gupta, and P. B. Fisher. 2005. Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature 433:73-77.
27. Sass, K. J., D. D. Spencer, J. H. Kim, M. Westerveld, R. A. Novelly, and T. Lencz. 1990. Verbal memory impairment correlates with hippocampal pyramidal cell density. Neurology 40:1694-1697.
28. Scheld, W. M., U. Koedel, B. Nathan, and H. W. Pfister. 2002. Pathophysiology of bacterial meningitis: mechanism(s) of neuronal injury. J. Infect. Dis. 186(Suppl. 2):S225-S233.
29. Sherry, D. F., L. F. Jacobs, and S. J. Gaulin. 1992. Spatial memory and adaptive specialization of the hippocampus. Trends Neurosci. 15:298-303.
30. Sousa Vde, O., L. Romao, V. M. Neto, and F. C. Gomes. 2004. Glial fibrillary acidic protein gene promoter is differently modulated by transforming growth factor-beta 1 in astrocytes from distinct brain regions. Eur. J. Neurosci. 19:1721-1730.
31. Stenzel, W., S. Soltek, D. Schluter, and M. Deckert. 2004. The intermediate filament GFAP is important for the control of experimental murine Staphylococcus aureus-induced brain abscess and Toxoplasma encephalitis. J. Neuropathol. Exp. Neurol. 63:631-640.
32. Tuomanen, E., B. Hengstler, R. Rich, M. A. Bray, O. Zak, and A. Tomasz. 1987. Nonsteroidal anti-inflammatory agents in the therapy for experimental pneumococcal meningitis. J. Infect. Dis. 155:985-990.
33. Vaher, P. R., V. N. Luine, E. Gould, and B. S. McEwen. 1994. Effects of adrenalectomy on spatial memory performance and dentate gyrus morphology. Brain Res. 656:71-78.
34. van der Flier, M., S. P. Geelen, J. L. Kimpen, I. M. Hoepelman, and E. I. Tuomanen. 2003. Reprogramming the host response in bacterial meningitis: how best to improve outcome Clin. Microbiol. Rev. 16:415-429.
35. Wellmer, A., C. Noeske, J. Gerber, U. Munzel, and R. Nau. 2000. Spatial memory and learning deficits after experimental pneumococcal meningitis in mice. Neurosci. Lett. 296:137-140.
36. Zhu, L., S. Ramboz, D. Hewitt, L. Boring, D. S. Grass, and A. F. Purchio. 2004. Non-invasive imaging of GFAP expression after neuronal damage in mice. Neurosci. Lett. 367:210-212.
37. Zysk, G., W. Bruck, J. Gerber, Y. Bruck, H. W. Prange, and R. Nau. 1996. Anti-inflammatory treatment influences neuronal apoptotic cell death in the dentate gyrus in experimental pneumococcal meningitis. J. Neuropathol. Exp. Neurol. 55:722-728.(Jagath L. Kadurugamuwa, K)