Ten male Long Evans rats, weighing 200–224 g at study onset, were housed on a 12:12 light/dark cycle. Food and water were available ad libitum. All experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee of the Cleveland Clinic.

Focal ischemia was induced as detailed previously (Cooperrider et al., 2014). Briefly, five rats were anesthetized with ketamine (50 mg/kg) and dexmedetomidine (0.5 mg/kg), fixed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA), and a craniotomy was performed over the sensorimotor cortex of the left cerebral hemisphere. Ischemia was induced by six intracortical injections of 800 pmol/2μl ET-1 (Millipore, MA, USA) at each of six stereotaxic coordinates in relation to bregma: (1) AP: −1.0, ML: +2.5, DV: −2.3; (2) AP: +1.0, ML: +2.5, DV: −2.3; (3) AP: +3.0, ML: +2.5, DV: −2.3; (4) AP: −1.0, ML: +3.5, DV: −2.3; (5) AP: +1.0, ML: +3.5, DV: −2.3; and (6) AP: +3.0, ML: +3.5, DV: −2.3 (Paxinos and Watson, 1998). After the injections were complete, the craniotomy was covered with cellulose paper (Data Sciences International) and tissue adhesive (Vetbond, 3M) to form a protective seal. Dexmedetomidine anesthesia was reversed with atipamezole (1 mg/kg), followed by buprenorphine (0.05 mg/kg, sc) administration for prophylactic pain management. The animals were monitored until fully recovered and returned, thereafter, to their home cages where food and water was provided ad libitum. Naïve control animals (n = 5) were used, as it is our experience that vehicle (i.e., saline injected) controls do no exhibit behavioral changes or cortical damage (Data not shown).

Six weeks after stroke induction, rats were anesthetized with 50 mg/kg sodium pentobarbital followed by decapitation. Brains were removed and fixed in 4% phosphate buffered paraformaldehyde (EMS, Hatfield, PA, USA) for 3 days. After cryoprotection in 30% sucrose/PBS, brain blocks were snap-frozen and preserved at −80°C until slicing by cryostat. Thirty micrometer sections of cerebral cortex and cerebellum were mounted on polysine-coated slides.

Cerebral and cerebellar sections were first stained with 0.4% cresyl violet in acidic PBS for 20 min after rehydrating with distilled water for 2 min. Cresyl violet staining was then destained by 70% ethanol in 1% acetic acid for 1 min. After dehydration with a series of ascending concentrations of ethanol and defatting with HistoClear, stained sections were mounted and coverslipped. Slices spanning the sensorimotor cortex as well as the cerebellum were mounted for analysis. Specifically, we mounted cortical sections corresponding to coordinates 4.2 mm anterior to bregma through 1.8 mm posterior to bregma, while coordinates for cerebellar sections were from 10.92 to 11.52 mm posterior to bregma (Paxinos and Watson, 1998). Stroke volumes were calculated as previously described by our group, utilizing a semi-automated method, SLICE (Park et al., 2013). Briefly, the lesion area of each Nissl-stained section was measured and the volume estimated by interpolation between the sections. The stroke lesions were then three-dimensionally projected onto the stereotactic atlas of the rat brain (Paxinos and Watson, 1998). Measurements were taken from both the stroke and naïve groups, despite the lack of significant change anticipated in the latter group.

The volume of the LCN (−10.92 mm to −11.52 mm to bregma), both ipsilesional and contralesional, was calculated using similar techniques. In order to be consistent with the stroke animals, the right LCN of the naïve cohort was defined as the index (pseudo-contralesional) location for the purpose of percent change calculations. Differences in LCN volume between the two hemispheres were calculated with the following equation:

The highly-packed nature of the cerebellar granule neurons at the cerebellar cortex makes quantification of cell density impossible. Instead, we quantified the thickness of the internal granule and molecular layers of Crus 1 of the ansiform (Crus 1) and PFL, both of which are located in the lateral cerebellum and the 4Cb, located in the vermis, as an index of cerebral cortical ET-1 induced changes. Under 100× magnification, six measurements of thickness, each 20 μm apart, were taken using ImageJ (RRID: SCR_003070, NIH) for each lobule, both contralesional and ipsilesional. Areas selected for measurement are shown in Figure 2A. The thickness of the cerebellar lobules was examined from −10.92 mm to −11.52 mm in the anterior/posterior plane (in relation to bregma). Data were expressed as mean thickness (μm) ± SEM and the change (%) in thickness was calculated with the following equation:

As above, data from the right cerebellar hemisphere of naïve animals were operationally defined as the “contralesional” side for the purpose of this calculation.

In order to evaluate apoptosis in the perilesional cerebral cortex, the cerebellar cortex and the LCN, cells were analyzed with a TUNEL assay kit (S7111, Millipore, MA, USA). Sections initially were incubated with TdT enzyme, which links digoxigenin-dNTP to apoptotic DNA fragments. Anti-digoxigenin antibody conjugated with fluorescein was applied to detect the digoxigenin-dNTP tails. After TUNEL assay, sections were counterstained with fluorescent Nissl dye (Life technologies, Carlsbad, CA, USA). TUNEL+/Nissl+ cells in the selected regions were counted by a blinded investigator at 200× magnification. The data were expressed as the density of TUNEL+/Nissl+ cells in a given area ± SEM.

To characterize cell loss in the LCN, the contralesional and ipsilesional LCN of each Nissl-stained cerebellar section was highlighted at ×100 magnification and the area of the LCN was measured by ImageJ software (NIH). Thereafter, the number of Nissl+ cells in the LCN was quantified by the same program. Density of Nissl+ cells in the highlighted LCN was calculated with the following equation:

Comparisons of stroke volume and percentage reduction of internal granule and molecular layers were analyzed by Student’s t-test. Other comparisons were analyzed by one-way ANOVA with post hoc Tukey test using GraphPad Prism software (RRID: SCR_002798, GraphPad, La Jolla, CA, USA).

ET-1 induced a significant lesion in the motor cortex of the stroke group (12.33 ± 0.18 mm³ vs. naïve: 0.194 ± 0.037 mm³, p < 0.0001, Figure 1A). Apoptotic cells (TUNEL+ cells) were observed in the perilesional area at a significantly higher rate than in the same topography on the contralesional motor cortex or in either cerebral hemisphere of the naïve group (Figure 1B). There was a significant increase of TUNEL+/Nissl+ cells in the ipsilesional motor cortex when compared to the contralesional motor cortex (One-way ANOVA: F_(3,82.78), p < 0.00001; Tukey’s test: ipsilesional: 10.23 ± 1.11 cells/mm² vs. contralesional: 0.13 ± 0.03 cells/mm²; p < 0.0001, Figure 1B) and naïve group (naïve, left motor cortex: 0.098 ± 0.02 cells/mm²; p < 0.0001, Figure 1B).

ET-1 administered at the cerebral cortex was associated with a significant reduction in the thickness of the contralesional cerebellar cortex. Specifically, there was a significant reduction in the thickness of the molecular layer at the Crus 1 (One-way ANOVA: F_(3,14.06), p < 0.0001; Tukey’s test, p < 0.0001; Figure 2B) and PFL (One-way ANOVA: F_(3,17.20), p < 0.0001, Tukey’s test, p < 0.0001; Figure 2C), but not 4Cb (One-way ANOVA: F_(3,0.70), p = 0.57, Tukey’s test, p = 0.24; Figure 2D) in the contralesional cerebellar cortex of stroke rats when compared to naïve rats. When compared to the ipsilesional sides of the same group, there were significant reductions in the thickness of the molecular at the Crus 1 (p < 0.01, Figure 2B) and PFL (p < 0.01, Figure 2C) in the stroke rats. However, there was no significant difference in 4Cb thickness (p = 0.88, Figure 2D). When comparing the percent reduction of thickness between the naïve and stroke groups the results were similar, with significant reductions of thicknesses of the molecular layer in Crus 1 (p < 0.0001, Figure 2E) and PFL (p < 0.0001, Figure 2E), but not in 4Cb (p = 0.33, Figure 2E).

At the level of the internal granule layer, comparing to the contralesional side of naïve rat, there was a significant reduction at the PFL (One-way ANOVA: F_(3,3.21), p < 0.05; Tukey’s test: p < 0.05; Figure 2G) but only a trend towards reduced thickness in Crus 1 that failed to reach significance (One-way ANOVA: F_(3,2.65), p = 0.08, Tukey’s test: p < 0.05; Figure 2F), but not 4Cb (One-way ANOVA: F_(3,0.27), p = 0.84, Tukey’s test, p = 0.57; Figure 2H). When comparing to the ipsilesional side of the same group, there were no significant reduction of thickness of internal granule layer in Crus 1 (p = 0.07, Figure 2F) and PFL (p = 0.06, Figure 2G). There was no difference detected in 4Cb (p = 0.97, Figure 2H). When comparing the percent reduction of internal granule layer thickness between stroke and naïve animals, there were significant reductions found in the Crus 1 (p < 0.0001; Figure 2I) and PFL (p < 0.0001; Figure 2I) but not 4Cb (p = 0.26, Figure 2I). No TUNEL+ cells were detected in either contralesional or ipsilesional cerebellar cortex of stroke animals.

ET-1-induced cerebral ischemia was associated further with significant reductions in the size of both the contralesional and ipsilesional LCN (Figure 3A). The contralesional LCN was reduced by 46% in stroke relative to naïve animals (One-way ANOVA: F_(3,30.79), p < 0.0001; Tukey’s test: stroke: 0.22 ± 0.05 mm³ vs. naïve: 0.41 ± 0.02 mm³; p < 0.0001, Figure 3B), while the ipsilesional LCN volume within the stroke group was reduced by 17% (stroke: 0.34 ± 0.02 mm³ vs. naïve: 0.41 ± 0.01 mm³; p < 0.05, Figure 3B).

The reduction of the LCN volume was accompanied by apoptosis in stroke animals (Figure 3C). The density of TUNEL+/Nissl+ cells in both contralesional (One-way ANOVA: F_(3,11.53), p < 0.0001; Tukey’s test: p < 0.0001, Figure 3D) and ipsilesional (p < 0.0001, Figure 3D) LCN in the stroke group was significantly higher than in naïve animals. Likewise, a within-subjects comparison of TUNEL+/Nissl+ cell density in the stroke group revealed significantly higher than in the ipsilesional LCN (p < 0.0001, Figure 3D).

There was a significant difference in cell density between the contralesional and ipsilesional LCN of animals in the stroke group (One-way ANOVA: F_(3,12.45), p < 0.0001, Tukey’s test: p < 0.05, Figure 3E) that was not present in the naïve group (p = 0.97, Figure 3E). Furthermore, there was a significant reduction in Nissl+ cell density at the contralesional LCN of the stroke group when compared to that of naïve (p < 0.0001, Figure 3E). This finding was consistent with the reduction of LCN volume.

Denervation of the CPC pathway leads to a reduction of excitatory input to the cerebellum and is the etiological factor thought to contribute to reduced neural activity along with diminished blood flow and, ultimately, neuronal death in the contralesional cerebellar cortex (Taylor et al., 2006; Gold and Lauritzen, 2002 #33; Jiménez-Caballero, 2012; Machado and Baker, 2012). The pontine nucleus primarily targets contralateral cerebellar structures; with projections diverging towards both the cerebellar cortex (Brodal and Steen, 1983; Brodal and Bjaalie, 1997; Xiong et al., 2002) and the deep cerebellar nuclei, including the LCN (Shinoda et al., 1992, 1993; Mihailoff, 1994). This distribution leaves both cortical and subcortical regions subject to deafferentation and degeneration in response to reduced cerebral input. At the level of the cerebellar cortex, we observed changes predominantly in more lateral cortical regions, with no significant reduction in the thickness across either the molecular or internal granule layers of 4Cb. This differential impact may simply reflect that, although the CPC pathway does project to the vermis (Xiong et al., 2002), the vast majority of its projections target the lateral cerebellum (Serapide et al., 2002). Volume changes in the LCN are similarly attributable to a loss of cortical input to the pontine nuclei. However given that the LCN also receives GABAergic Purkinje cell input (Tsubota et al., 2013) from the degenerating cerebellar cortex, the precise role of loss of excitatory (from the pontine nuclei) vs. inhibitory (from the cerebellar cortex) inputs on LCN degeneration is unclear. Further investigation of the afferent axons and synapses to the LCN is necessary to clarify the extent of contribution from each mechanism. Finally, while the atrophic changes were found to be more profound in the cerebellar hemisphere opposite the unilateral cerebral infarct, similar, albeit more modest, changes in the ipsilateral cerebellum likely underscore the bilateral nature of pontine projections (Cicirata et al., 2005). Notably, a limitation of the current study is that cerebral infarcts were generated only in the left hemisphere of animals and future studies should consider targeting each cerebral hemisphere in order to confirm that pattern of CCA reverses.

A more complete characterization of CCD/CCA pathology will help better understand long-term clinical findings after cortical ischemia and will be relevant to the development and interpretation of future preclinical experiments (Carrera and Tononi, 2014). Furthermore, the presence of CCD has been shown to limit the prognosis of post-stroke rehabilitation (Takasawa et al., 2002), suggesting that therapies should be aimed not only at facilitating changes in the perilesional cortex but also at reversing diaschisis (Machado and Baker, 2012). Following injury, the infarcted cerebral cortex may be re-organized under the influence of excitatory input from the DTC pathway. However, if the DTC is impaired at its origin (the LCN) as a result of CCA, there will be limited excitatory input to the cortex, potentially limiting plasticity and recovery. Novel treatment approaches, including the upregulation of neural activity across the DTC pathway, may facilitate reorganization and recovery (Machado et al., 2009, 2013; Cooperrider et al., 2014; Park et al., 2015). Overall, our current findings support the need to better characterize the nature and extent of cerebellar changes in the cortex and deep nuclei in response to cerebral infarct, both with regards to the mechanistic underpinnings of CCD and CCA as well as the potential to explore these phenomena for the development of novel therapies.