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. 2009 Aug;175(2):668-84.
doi: 10.2353/ajpath.2009.081126. Epub 2009 Jul 16.

Apoptosis of hippocampal pyramidal neurons is virus independent in a mouse model of acute neurovirulent picornavirus infection

Eric J Buenz  1 , Brian M Sauer, Reghann G Lafrance-Corey, Chandra Deb, Aleksandar Denic, Christopher L German, Charles L Howe
Affiliations

Apoptosis of hippocampal pyramidal neurons is virus independent in a mouse model of acute neurovirulent picornavirus infection

Eric J Buenz et al. Am J Pathol. 2009 Aug.

Abstract

Many viruses, including picornaviruses, have the potential to infect the central nervous system (CNS) and stimulate a neuroinflammatory immune response, especially in infants and young children. Cognitive deficits associated with CNS picornavirus infection result from injury and death of neurons that may occur due to direct viral infection or during the immune responses to virus in the brain. Previous studies have concluded that apoptosis of hippocampal neurons during picornavirus infection is a cell-autonomous event triggered by direct neuronal infection. However, these studies assessed neuron death at time points late in infection and during infections that lead to either death of the host or persistent viral infection. In contrast, many neurovirulent picornavirus infections are acute and transient, with rapid clearance of virus from the host. We provide evidence of hippocampal pathology in mice acutely infected with the Theiler's murine encephalomyelitis picornavirus. We found that CA1 pyramidal neurons exhibited several hallmarks of apoptotic death, including caspase-3 activation, DNA fragmentation, and chromatin condensation within 72 hours of infection. Critically, we also found that many of the CA1 pyramidal neurons undergoing apoptosis were not infected with virus, indicating that neuronal cell death during acute picornavirus infection of the CNS occurs in a non-cell-autonomous manner. These observations suggest that therapeutic strategies other than antiviral interventions may be useful for neuroprotection during acute CNS picornavirus infection.

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Figures

Figure 1
Figure 1
Magnetic resonance imaging reveals time-dependent development of hippocampal damage during acute TMEV infection of the CNS. T2-weighted imaging in two separate mice (A–F and G–L) at 0 (A,G), 1 (B,H), 2 (C,I), 3 (D,J), 4 (E,K), and 7 (F,L) days postinfection (dpi) revealed a time-dependent increase in hyperintense abnormalities located predominantly in the hippocampus. M and O: Hippocampal pathology by H&E staining at 7 dpi associated with MRI hyperintensity in the same mice in (N) and (P), respectively. The arrowheads delineate the regions of pathology (M,O) and hyperintensity (N,P) that are restricted to the CA1 pyramidal neuron layer in the hippocampus. Note that the T2 hyperintensity distribution closely follows the extent of pathology. Q and R: Rapid acquisition with relaxation enhancement imaging protocol that confirms the presence of hippocampal injury that increases from 1 dpi (Q) to 7 dpi (R). Arrowheads in Q and R represent extent of CA1 region in the hippocampus.
Figure 2
Figure 2
Magnetic resonance spectroscopy indicates hippocampal neuronal injury during acute TMEV infection of the CNS. Spectroscopic analysis of N-acetylaspartate (NAA) and creatine levels within a 27 μl voxel (D) containing the dorsomedial aspect of the hippocampus at 1 dpi (A) and 7 dpi (B) indicated a suppression of the NAA line relative to creatine. C: Quantitation of the ratio of NAA to creatine showed a 36% reduction consistent with neuronal injury in the hippocampus: t(4) = 6.181, P = 0.003 for 7 dpi vs 1 dpi by t-test.
Figure 3
Figure 3
Acute infection with TMEV results in damage localized to the hippocampus. Histological analysis by H&E staining indicated focal tissue damage isolated to the pyramidal neurons in the CA1 subfield of the hippocampus (B) at 7 dpi. Pathology was never observed in CA1 in sham-infected mice (A). Arrowheads delineate CA1 in (A) and (B). Scale bar = 500 μm in (B) (also refers to A).
Figure 4
Figure 4
Acute infection with TMEV results in damage localized to the hippocampus. A high resolution montage of the whole brain stained with Nissl, collected in the sagittal plane at 1 mm lateral to the midline at 7 dpi (A). Overt pathology is only observed in the hippocampus. B: Higher magnification indicates that hippocampal pathology at 7 dpi was restricted to the CA1 layer. Scale bars: 1 mm (A); 250 μm (B).
Figure 5
Figure 5
Acute infection with TMEV results in damage localized to the hippocampus. Serial, Nissl-stained sagittal sections collected from 0.3 mm lateral to the midline through to 3.6 mm lateral to midline (D). We observed no pathology at any level in the cerebellum (A), midbrain (B), or brainstem (C). Scale bars = 500 μm.
Figure 6
Figure 6
Acute infection with TMEV results in damage localized to the hippocampus. Serial, Nissl-stained sagittal sections collected from 0.25 mm lateral to the midline through to 4 mm lateral to midline show extensive damage to the entire medial extent of CA1 in the hippocampus (A) but no injury to cortex (B). Scale bars = 500 μm.
Figure 7
Figure 7
The majority of CA1 pyramidal neurons are lost by 7 dpi. Hippocampal sections at −1.7 mm from Bregma were collected from uninfected mice (A) and mice at 7 dpi (B and C) and stained for the neuron-specific marker NeuN. Montages of images collected at ×60 reveal that the high density of CA1 neurons present in uninfected controls (A) is attenuated following infection (B and C). D: Individual fields were subjected to an automated thresholding and counting routine to determine the number of NeuN-positive cells in CA1. While greater than 90 NeuN-positive cells were found per field in uninfected mice (n = 20 fields from 5 mice), fewer than 20 NeuN-positive cells were observed per field in mice at 7 dpi (n = 20 fields from 5 mice), and many animals had CA1 fields devoid of NeuN-positive structures. Scale bar in (C) = 100 μm; refers to A–C.
Figure 8
Figure 8
TMEV infection triggers a time-dependent injury to the pyramidal layer (stratum pyramidale) of the CA1 region of the hippocampus. A, C, E, G, I, K: Low magnification H&E image of the hippocampus. B, D, F, H, J, L: Higher magnification image collected within a portion of CA1 chosen to highlight representative pathology. A and B: Sham-infected animals exhibit normal hippocampal morphology 7 days after infection. The CA1 pyramidal layer is indicated by arrowheads in (A). The CA3 pyramidal layer is indicated by arrows in (A). DG: dentate gyrus. Normal CA1 pyramidal neuron apical dendrites are marked by an arrowhead in (B). C and D: No damage was evident at 1 dpi. E and F: By 2 dpi, pyramidal neuron damage near the CA1 and CA3 junction was detectable (arrowhead in E). Higher magnification of the damaged area revealed condensed neurons, vacuolization in the pyramidal layer architecture (arrow in F), and complete loss of the large apical dendrites (arrowhead in F). G and H: This process continued at 3 dpi, with the injury spreading medially along CA1 (arrowhead in G). I and J: By 4 dpi the majority of neurons in CA1 were injured or absent. K and L: By 7 dpi there was frank destruction of the hippocampal architecture surrounding CA1 but remarkable preservation of CA3 (delineated by arrowheads in K) and the dentate gyrus. Scale bar in (K) = 500 μm; refers to A, C, E, G, I, and K. Scale bar in (L) = 50 μm; refers to B, D, F, H, J, and L. These results are representative of at least 10 mice in each of three separate experiments.
Figure 9
Figure 9
Virus antigen is not restricted to the hippocampus and only a minority of CA1 neurons are infected. The distribution of TMEV at 2 dpi was determined by immunohistochemistry using a rabbit anti-TMEV polyclonal antibody. Scattered populations of infected cells were detected in the cortex (black arrows in A and B), thalamus (arrowheads in A and B), fimbria-fornix (gray arrows in A), hippocampus (gray arrows in B), and brainstem (arrows in C). Within the hippocampus, the distribution was highly variable, with concentrations of cells at the CA1:CA3 border (D). Higher magnification analysis of the hippocampus from a different mouse revealed that the majority of CA1 neurons were uninfected (E). Images are representative of at least five animals. Scale bars: 2 mm (C; refers to A–C); 500 μm (D); 100 μm (E).
Figure 10
Figure 10
EM evidence that hippocampal pyramidal neurons undergo apoptosis during acute TMEV infection. Electron microscopy was used to examine cellular morphology in the CA1 layer of the hippocampus. Hippocampal thick sections were collected at 4 dpi from sham-infected (A,C,E) and TMEV-infected (B,D,F) mice and stained with Toluidine Blue O to identify the CA1 region. Thin sections of CA1 were then prepared for EM analysis. Note the considerable difference in pyramidal cell layer architecture and the apoptotic morphology of pyramidal neurons between sham- (A) and TMEV-infected (B) animals at low magnification. Higher magnification showed the morphology of normal pyramidal neurons in sham-infected mice (C,E). In sections from TMEV-infected mice (D,F) the majority of CA1 neurons showed a shrunken morphology, cellular blebbing (arrow in F), and chromatin condensation along the nuclear membrane (arrowhead in F), consistent with apoptosis. In addition, vacuolization of the area surrounding the dying neuron (D,F) was consistent with the vacuolization seen by histological analysis and light microscopy. Scale bars: 20 μm (in B and applies to A); 5 μm (D and applies to C); 2.5 μm (E); 2.5 μm (F).
Figure 11
Figure 11
Acute TMEV infection induces DNA fragmentation specifically within the CA1 pyramidal layer of the hippocampus. TUNEL staining revealed considerable DNA fragmentation within cells localized almost exclusively to the CA1 region of the hippocampus at 4 dpi (blue = DAPI; green = TUNEL). Sham-infected animals exhibited essentially no TUNEL-positive cells anywhere in the brain (A and B), while mice infected with TMEV showed a large number of cells with DNA fragmentation localized to the CA1 region of the hippocampus at 4 dpi (C and D). Quantitation of the number of TUNEL-positive cells in the hippocampus revealed a significant difference (P = 0.002 by the Mann-Whitney rank sum test) between sham-infected (22 ± 8 TUNEL-positive cells per hippocampal section; n = 6) and TMEV-infected (530 ± 142 TUNEL-positive cells per hippocampal section; n = 6) animals. The size of the hippocampal area analyzed did not differ between the groups. Scale bars: 500 μm (C and applies to A); 500 μm (in D and also applies to B).
Figure 12
Figure 12
Death of CA1 pyramidal neurons is independent of direct infection with the TMEV virus. Colocalization of TUNEL immunostaining (A; green in C) and TMEV antigen immunostaining (B; red in C) revealed the presence of many dying neurons that were not infected with the virus (green-only cells in C) at 4 dpi. This observation was confirmed by the extensive absence of colocalization between TUNEL immunohistochemistry (D; pseudocolored green in F) and in situ hybridization for viral RNA (grains in E; pseudocolored red in F) at 4 dpi. While several cells are both infected and dying (yellow in F), the majority of dying neurons are not infected (green-only in F). DAPI is shown in blue in (C). Scale bar: 50 μm (C and refers to A–C); 50 μm in (F and refers to D–F).
Figure 13
Figure 13
Hippocampal injury during acute TMEV infection is associated with apoptotic markers. Higher magnification images of the same areas of CA1 analyzed in Figure 8. A, D, G, J, M, P: Oxidative damage assessed by anti-8-OHdG immunostaining (red). B, E, H, K, N, Q: Activated caspase-3 immunoreactivity (red). C, F, I, L, O, R: TUNEL staining to show DNA cleavage (green). A–C: The CA1 region of sham-infected animals shows no evidence of oxidative damage (A), activated caspase-3 (B), or TUNEL positivity (C) at 7 dpi. D–R: In contrast, mice infected with TMEV show a time-dependent increase in oxidized nucleotides, activated caspase-3, and DNA cleavage. The arrow in (M) indicates a typical anti-8-OHdG-positive pyramidal neuron. The arrowhead in (K) indicates a typical activated caspase-3-positive pyramidal neuron. The hand in (O) indicates a typical TUNEL-positive pyramidal neuron. The day of maximum caspase-3 activation (H or K) precedes the onset of maximum DNA cleavage (O and R), while the onset of oxidative damage (G) is contiguous with caspase-3 activation. Scale bar = 50 μm (in R and applies to all panels). These results are representative of at least five mice in each of three separate experiments. DAPI-labeled nuclei are blue in all panels.
Figure 14
Figure 14
Cell death in the hippocampus involves activated caspase-3 and activated calpain. A: Hippocampi were freshly isolated from mice (n = 3 at each time point) at the indicated days postinfection and caspase-3 and calpain-1 activity was measured using a cell-free substrate cleavage assay (A). Both caspase-3 and calpain-1 exhibited a peak in activity at 3 dpi. B: Immunoblots of total (top band) and cleaved (lower band) β spectrin indicate an accumulation of this calpain-1 cleavage product during the course of infection, indicative of calpain-1 activation. C: Immunoblots of activated caspase-3 confirm the substrate cleavage data. The immunoblot analyses are representative of two separate experiments.
Figure 15
Figure 15
Mice exhibit memory defects following acute infection with TMEV. Two weeks after infection, mice were tested in the Morris water maze using a competition test strategy to determine whether reference memory was disrupted. In this test, after 1 week of training and acquisition testing, the submerged training platform (small dark gray circles in C) is removed and a visible competition platform is placed in the tank in the opposite quadrant (light gray circles in C). If mice have developed a spatial map of the maze they ignore the visible platform and search in the training quadrant. In contrast, the absence of a spatial memory map results in direct escape to the visible platform. Sham-infected mice (n = 8) exhibited clear evidence of spatial memory formation, with significantly longer escape latency (A) and significantly more time spent in the empty training quadrant (B) than TMEV-infected mice (n = 8). As visual confirmation of this difference, swim paths are shown for three mice in each group (C). The sham-infected animals search the training quadrant before escaping via the visible platform. The TMEV-infected mice swim directly to the visible platform. Likewise, mice were tested at 7 dpi after infection in a scent-based novel object recognition test (D and E). In this test, mice are individually habituated to an environment consisting of a 30 cm × 30 cm acrylic box lined with wood shavings (changed between each mouse to eliminate spurious olfactory cues). After 5 minutes, the mice are briefly removed to a holding cage, and then reintroduced to the testing environment containing two identical scent objects. Testing of numerous objects revealed that apple-, berry-, and cherry-scented tea candles induced considerable interrogation but did not stimulate feeding behavior. During the 10-minute training session mice were exposed to 2 berry-, 2 apple-, or 2 cherry-scented candles (white circles in D), then removed to the holding cage for 5 minutes. The testing phase consisted of reintroducing the mice to the environment containing one candle from the training session and one new candle of a different scent (the black circle in D), randomly assigned. Mice were video taped and the number of interrogations of each object was determined manually during the training and testing sessions. The ratio of interrogations of the novel object to the number of interrogations of the familiar object was taken as a discrimination index. During training, all groups exhibited a discrimination index of 1 (dashed line in E). During testing, uninfected controls (n = 13 mice) exhibited an increase in the discrimination index consistent with interrogating the novel object two times more than the familiar object. In contrast, mice lesioned with kainic acid (n = 4 mice) or mice at 7 dpi (n = 12 mice) exhibited a discrimination index of 1, consistent with an inability to recall the familiar object.
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