Hematopoietic cell activation in the subventricular zone after Theiler's virus infection

Eighty 6–7 week old female wild type SJL/J mice (Taconic Labs) were used in the TMEV studies. All mice were housed in the Northwestern University animal care containment facility and were provided with unlimited access to standard laboratory food and water. Easier access to food and water was provided for TMEV injected animals exhibiting neurological impairment.

Three groups of mice were used: TMEV, sham, and naïve. Together, sham and naïve mice constituted "controls". TMEV and sham mice were anesthetized with 4% isoflurane. 3 × 10⁶ PFU BeAN 8386 virus, suspended in sterile 0.03 ml BSS, was injected into the right cerebellar cortex through a 27 gauge needle fitted with a needle guard to prevent penetration beyond 3.5 mm ventral to skull. Injections were localized to a point half-way to midline at ear level. Sham mice received injections of 0.03 ml BSS. Naïve mice were not anesthetized or injected. All mice were monitored for changes in neurological status two to three times per week. Mice were assigned numerical scores as follows: 0 = asymptomatic; 1 = mild waddling gait; 2 = moderate waddling gait without spastic paralysis; 3 = severe waddling gait with mild spastic paralysis; 4 = severe waddling gait with moderate to severe spastic paralysis; 5 = total hind limb paralysis; 6 = moribund. Mice were further divided into three groups according to clinical scores or time point match for sham and naive: preclinical = prior to onset of any symptoms but after initial inflammatory/increased stress due to injections (D 14–24); early onset = clinical score of 1 or more for 2 consecutive days (D 42–47); chronic = increased clinical score (2 or 3) for minimum of 5 consecutive days (D 90). Each group had its respective control mice. Preclinical sham N = 4, Preclinical naïve N = 1, Preclinical TMEV N = 27; early onset sham N = 5, early onset naïve N = 1, early onset TMEV N = 25; chronic sham N = 4, chronic naïve N = 1, chronic TMEV N = 4.

Mice were perfused with 4% paraformaldehyde, brains post-fixed overnight, and cryoprotected in 30% sucrose overnight at 4°C before sectioning. Free-floating coronal sections, 30 μm thick, were cut on a sliding microtome and stored in cryoprotectant at -20°C. Antibodies used: rat anti-CD45 (clone IBL-5/25; 1:500, Chemicon, Temeluca, CA); goat anti-doublecortin (C-terminus; 1:200, Santa Cruz Biotechnology, Santa Cruz California), rabbit anti-BeAn (1:600, Miller Lab, Northwestern University), mouse anti-PSA-NCAM (1:500, Chemicon), rabbit anti-phosphohistone3 (1:500, Upstate Biotechnology). Sections were washed and blocked with 50 mM glycine in phosphate buffered saline (PBS) to reduce autofluorescence of paraformaldehyde-fixed tissue. Sections were washed, blocked in PBS containing 0.1% Triton X-100 and 10% Donkey Serum, DS (Sigma), (PBS+), incubated overnight at 4°C in primary antibodies, washed, incubated one hr at RT in Cy2 (1:200) or Cy3 (1:500) conjugated anti-primary secondaries, and rinsed in phosphate buffer (PB). Sections were mounted and slides were coverslipped with FluorSave mounting medium (Chemicon) in PBS. Ommission of primary antibody was used as controls in all immunohistochemistry experiments.

A comprehensive anterior to posterior set of brain sections were examined at the following anatomical coordinates [33]. Olfactory bulb (4.0 to 3.0 mm anterior to bregma), anterior cortex (3.0 to 2.0 mm anterior to bregma), striatum (1.7 to -0.5 mm from Bregma), hippocampus (-1.0 to -2.5 mm posterior to bregma), cerebellum (-5.6 to -7.0 mm from bregma). In addition, we collected sections from cervical, thoracic, and lumbar spinal cord. Immunohistochemistry was examined and recorded on a Leica DMIRB microscope using Openlab software and on a Zeiss Meta confocal microscope, and analyzed in 3-D with Zeiss and Volocity Software. Images were composed in Adobe Photoshop. Doublecortin SVZ neurogenesis quantification. Doublecortin Cell Counts. Images of doublecortin and DAPI labeled sections of the dl SVZ were taken at 63× on a Zeiss Meta confocal microscope with a single scan of both channels. The images were saved and exported as tiff images into Openlab Image software (Improvision). In Openlab Dcx+ cells and DAPI+ cell nuclei in the dl SVZ were counted. Dcx immunofluorescence surface area measurements. We used these measurements to confirm Dcx+ cell counts. Images of doublecortin labeled sections were taken at identical camera settings and positive Dcx immunofluorescence intensity threshold levels pre-determined. We used these levels to calculate the percent of the SVZ surface area occupied by positive Dcx immunofluorescence in the dorsolateral SVZ (dl SVZ). Hippocampal Dcx+ cell counts. All Dcx+ cells were counted in the subgranular zone of the dentate gyrus unilaterally. Emigrated Dcx+ cell quantification. The large majority of cells that emigrated in controls and after TMEV did not move more then a few hundred microns from the SVZ. Therefore the large majority of cells were within the distances sampled. Doublecortin labeled striatal sections were viewed at 40× on a Leica DMIRB upright microscope. Emigrated Dcx+ cells were counted unilaterally. With the SVZ to the left margin of the field of view at 40×, Dcx+ cells in the striatum were counted, with the SVZ to the right margin of the field of view, septal Dcx+ cells were counted, with the SVZ in the middle of the field showing the full extent of the CC thickness, Dcx+ cells in the CC were counted, with the ventral 3rd of the SVZ in the middle of the field, emigrated Dcx+ cells in the ventral 3rd septum, striatum and surrounding ventral SVZ were counted. Phosphohistone3 cell counts. Images of phosphohistone3 labelled sections of the dorsolateral, medial septal and ventral-medial SVZ and were taken at 40× on a Leica DMRIB microscope. The images were saved and exported as tiff images into Volocity (Improvision). The number of phosphohistone H3+ cells were counted and averaged for 6 consecutive sections per animal ranging from ~bregma 0.9 to 0.0. Only cells that had bright, complete labelling in the nuclei were included, cells that showed fragmented staining were excluded. All measurements were taken by staggering controls and treated mice to avoid quantification drift bias. Satistics were performed in Microsoft Excel; Student's T-tests (two tailed, equal variance) with p values < 0.05 were considered to be significant.

TMEV was injected in mice into the left cerebellar cortex (Fig. 1A). After infections, mice were sacrificed at 14–24 days (preclinical, PC), 42–47 days (early onset, EO), and 90 days (Chronic, C) (Fig. 1B), based on behavioural criteria as described in the Methods section. Inflammation and hematopoietic cell activation was scored semi-quantitatively using a 4 point scale based upon cell morphology, cell density, and brightness of CD45+ immunofluorescence (Fig. 1C,D). This was done primarily to allow a within-study comparison of hematopoietic cell activation across regions and time. 0: weakly labeled CD45+ cells (CD45^low) with highly ramified processes. CD45+ cells are evenly distributed throughout parenchyma. 1: CD45^low microglia became CD45^medium or CD45^high and changed from highly ramified to cells with larger soma and shorter, thicker processes. A score of 1 was characterized by relatively few round CD45^high cells that were dispersed randomly. 2: intermittent focal areas of high density CD45^high, round cells. 3: multiple focal and/or contiguous areas of high density CD45^high, round cells, with or without large, irregularly shaped cells. Each stage showed a corresponding decrease of the previous stage's morphology and relative CD45+ intensity (Fig. 1C,D).

In examining CD45+ cell activation, we noted that the specific location varied from mouse to mouse, and from section to section. In contrast, CD45+ activation was very consistent in the SVZ and immediately surrounding regions (Figs. 5, 6). Unlike other brain regions that normally exhibit CD45^low levels, SVZ cells seem to be constitutively "semi-activated": they have ramified processes and express CD45 at medium (and occasionaly high) levels [7] (Fig. 5C,G). At preclinical TMEV stages, most CD45+ SVZ cells became activated; they were round and CD45^high (Fig. 5B,D,H). CD45^high cells in and around the SVZ were frequently associated with blood vessels that had perivascular cuffs of activation. SVZ CD45^high cells were seen the length of the lateral ventricles, and were prominent in the ventral SVZ (Fig. 2, Fig. 5B, Fig. 6) an area that exhibits a high degree of neurogenesis [36]. Though CD45+ cell activation in the SVZ was present at all time points, the preclinical group had greatest activation and the chronic group the least (Fig. 5D–F, Fig. 6). Interestingly, CD45+ cell activation was also consistently high in the rostral migratory stream as it courses through the anterior cerebral cortex into the olfactory bulb (Fig. 2). Similar to the SVZ, this was characterized by round CD45^high cells and was most prominent in preclinical mice.

CD45 cell activation also consistently occurred in regions close to the SVZ; the dorsal septum and striatum. Similar to the rest of the forebrain, this occurred most prominently in preclinical mice, but was also apparent in early onset and chronic mice (Fig. 2, Fig. 5B, Fig. 6). The corpus callosum, immediately dorsal to the SVZ, also contained activated CD45+ cells however, to a lesser extent than grey matter areas (Fig. 5B). We next examined whether consistent CD45+ cell activation occurred in the other neurogenic region of the forebrain, the dentate gyrus of the hippocampus. The majority of activity was in the dorsal hippocampus subjacent to the lateral ventricle, sandwiched between it and the corpus callosum (Fig. 2, 5I,J). CD45+ cell activation was evident in the CA1, CA2, pyramidal and oriens layers of the hippocampus, however it was notably absent from the dentate gyrus (Fig. 2, 5I,J). Nearby, CD45^high cell activation was observed in the dorsal fimbria and consistently in the 3^rd ventricle (Fig. 2, Table 1).

TMEV infects macrophages but has recently been proposed to target polysialic acids [26, 37]. The SVZ is the forebrain region with the highest concentration of polysialylated neural cell adhesion molecule (PSA-NCAM), which is specifically expressed by SVZ neuroblasts [38, 39]. Thus, we queried whether TMEV infects macrophages and SVZ neuroblasts, and thereby concentrates the viral load in periventricular regions. Immunohistochemistry against the BeAn strain of virus revealed that the virus was found in and around the SVZ in regions of high CD45 activation (Fig. 7A). We immunostained for BeAn and CD45 as well as doublecortin (Dcx), a marker of SVZ neuroblasts [36, 40]. Virus was associated with both CD45+ cells (not shown) and SVZ neuroblasts (Fig. 7A–C). Fig. 7D, a Z-stack of high magnification confocal microscopy optical sections shows close association between BeAn immunoreactivity and a Dcx+ neuroblast in the SVZ.

We next asked whether the massive CD45 cell activation in the SVZ was general to demyelinating injuries. Cuprizone, a copper chelating agent induces demyelination in a variety of CNS regions including immediately above the SVZ, in the corpus callosum [41, 42]. As expected based on known patterns of demyelination after cuprizone [43], CD45+ cells in the corpus callosum were highly activated after cuprizone (Fig. 8A,B), however, CD45 expression and cell numbers in the SVZ and periventricular striatum remained at control levels (Fig. 8C,D). This result suggests that the TMEV induced activation in the SVZ and periventricular regions is not a general response to demyelination.

We next examined if SVZ CD45+ cell activation after TMEV infection was associated with changes in neuroblast numbers or with SVZ emigration. Striatal and hippocampal sections were labeled with anti-doublecortin (Dcx) antibodies. Dcx is a microtubule associated protein expressed by SVZ neuroblasts and is accepted as an effective read-out for SVZ neurogenesis and emigration [36, 40, 44, 45]. In control mice, Dcx+ neuroblasts are organized in tight arrays of cells [46, 47] (Fig. 9A). In infected mice, SVZ neuroblast cell-cell contacts were disrupted (Fig. 9B,C) and their membranes exhibited a loss in integrity leading to an amorphous morphology in some cells. This was especially apparent in sections that had the highest amount of CD45+ cell activation. Similar results were found with another major marker of SVZ neuroblasts; polysialylated neural cell adhesion molecule (PSA-NCAM) [38, 39]. Compared to sham mice, which had contiguous arrays of PSA-NCAM+ chains, preclinical mice had significant gaps in between these chains (Fig. 9H,I). Although our results at the light microscopic level suggest loosening of neuroblast-neuroblast contacts, ultimate determination of this finding would require analysis at the electron microscope level.

Dcx immunofluorescence, Dcx+ cell numbers, and DAPI+ nuclei in the SVZ were quantified (Methods) but were not changed significantly in preclinical and early onset mice. We examined proliferation directly in the SVZ with immunohistochemistry for the G2/M cell cycle marker phosphohistone3 (PH3) [48] and found that numbers of PH3+ cells in the SVZ were not different between controls and TMEV infected preclinical and early onset mice. Though the number of Dcx+ cells, and the percent surface area occupied by Dcx immunfluorescence in the SVZ decreased at late time points, the intensity of Dcx immunofluorescence did not change appreciably at any time point. Interestingly, the number of Dcx+ cells was significantly decreased in chronic mice (38 ± 5 vs. 23 ± 5, p = 0.009), although the total number of cells (DAPI+) remained similar. This suggests that the number of Dcx+ cells decreased at late time points rather then simply the level of Dcx expression decreasing. Similar results were obtained with PSA-NCAM, with the intensity of immunofluorescence remaining similar but the surface area occupied by PSA-NCAM+ cells decreasing in the SVZ. In the hippocampal dentate gyrus (DG), there was no significant difference in Dcx+ cell counts between control and experimental animals at the first two time points. However, similar to the SVZ, there was also a statistically significant loss of DG Dcx+ cells in chronic experimental animals (24 ± 3 vs. 10 ± 2, p = 0.0007).

Small numbers of Dcx+ cells constitutively emigrate dorsally into the corpus callosum and ventrally into the nucleus accumbens and striatum [36]. Increased numbers of Dcx+ SVZ neuroblasts emigrate after a variety of insults such as stroke [13] and traumatic brain injury [49]. However it was not known if neuroblasts also emigrate into ectopic areas after TMEV infection. We found that Dcx+ cells emigrating as individuals, or in clusters of cells, were increased in mice at the time of the highest CD45+ cell activation in the SVZ (Fig. 9D–G). Preclinical experimental animals showed increased emigration into the septum, striatum, and accumbens (Fig. 9D–G; Fig. 10). In contrast, the number of Dcx+ cells in the corpus callosum were not increased (Fig. 9D; Fig. 10). By the early onset time point, numbers of emigrated cells returned to normal (Fig. 9A–C; Fig. 10). Unexpectedly, the numbers of ventrally emigrating cells was slightly decreased in chronic mice (Fig. 9C; Fig. 10), matching the loss of Dcx+ cell numbers in the SVZ at that time point. These results suggests that TMEV causes SVZ neuroblasts to emigrate into grey matter at preclinical stages of the disease.