Cell based therapy enhances activation of ventral premotor cortex to improve recovery following primary motor cortex injury

Abstract

Stroke results in enduring damage to the brain which is accompanied by innate neurorestorative processes, such as reorganization of surviving circuits. Nevertheless, patients are often left with permanent residual impairments. Cell based therapy is an emerging therapeutic that may function to enhance the innate neurorestorative capacity of the brain. We previously evaluated human umbilical tissue-derived cells (hUTC) in our non-human primate model of cortical injury limited to the hand area of primary motor cortex. Injection of hUTC 24 h after injury resulted in significantly enhanced recovery of fine motor function compared to vehicle treated controls (Moore et al., 2013). These monkeys also received an injection of Bromodeoxyuridine (BrdU) 8 days after cortical injury to label cells undergoing replication. This was followed by 12 weeks of behavioral testing, which culminated 3 h prior to perfusion in a final behavioral testing session using only the impaired hand. In this session, the neuronal activity initiating hand movements leads to the upregulation of the immediate early gene c-Fos in activated cells. Following perfusion-fixation of the brain, sections were processed using immunohistochemistry to label c-Fos activated cells, pre-synaptic vesicle protein synaptophysin, and BrdU labeled neuroprogenitor cells to investigate the hypothesis that hUTC treatment enhanced behavioral recovery by facilitating reorganization of surviving cortical tissues. Quantitative analysis revealed that c-Fos activated cells were significantly increased in the ipsi- and contra-lesional ventral premotor but not the dorsal premotor cortices in the hUTC treated monkeys compared to placebo controls. Furthermore, the increase in c-Fos activated cells in the ipsi- and contra-lesional ventral premotor cortex correlated with a decrease in recovery time and improved grasp topography. Interestingly, there was no difference between treatment groups in the number of synaptophysin positive puncta in either ipsi- or contra-lesional ventral or dorsal premotor cortices. Nor was there a significant difference in the density of BrdU labeled cells in the subgranular zone of the hippocampus or the subventricular zone of the lateral ventricle. These findings support the hypothesis that hUTC treatment enhances the capacity of the brain to reorganize after cortical injury and that bilateral plasticity in ventral premotor cortex is a critical locus for this recovery of function. This reorganization may be accomplished through enhanced activation of pre-existing circuits within ventral premotor, but it could also reflect ventral premotor projections to the brainstem or spinal cord.

Keywords: Cell based therapy; Cortical damage; Functional recovery; Macaca Mulatta; Reorganization; Subgranular zone; Ventral premotor cortex; hUTC (Human umbilical tissue derived cells).

Figure 1. Regions of interest for stereological estimation of c-Fos and synaptophysin

The entire extent of dorsal and ventral premotor cortices were evaluated. Schematic representations of rhesus monkey coronal sections adapted from Paxinos (2008) are shown from rostral to caudal (A–H) showing the boundaries of the dorsal and ventral premotor area regions of interest that were sampled for stereological analysis. The sagittal view (I) shows the rostral and caudal boundaries from which the sections sampled were taken. Abbreviations: sar, superior arcuate sulcus. ps, principle sulcus. arsp, arcuate sulcus spur.

Figure 2. Regions of interest for stereological estimation of BrdU

The subventricular zone and granule cell layer of the dentate gyrus were evaluated. The boundaries of the subventricular zone ROI that was sampled for stereological analysis are shown as representative micrographs (A, C, E, G) and schematic illustrations (B, D, F, H). The boundaries of the dentate gyrus, including the granule cell layer that was sampled for stereological analysis, are shown as representative micrographs (I, K, M, O) and schematic illustrations (J, L, N, P). Abbreviations: L, Lateral Ventricle. SVZ, Subventricular Zone. ECL, Ependymal Cell Layer. SGZ, Subgranular Zone. ML, molecular layer. H, Hilus.

Figure 3. Differential activation of c-Fos positive cells

Representative micrographs of c-Fos activation in ipsilesional ventral premotor cortex in placebo (A) and hUTC treated (B) sections are shown. At a higher magnification, individual c-Fos positive cells of differential activation levels are discernable in placebo (C) and hUTC treated (D) sections. An example of a c-Fos labeled cell is shown with the black arrow in D. Scale bar: 100 μm

Figure 4. c-Fos positive cells in Ventral Premotor Cortex increase following hUTC treatment

Numbers of c-Fos positive cells in the dorsal and ventral premotor cortex were estimated using unbiased stereology. There were significantly more c-Fos positive cells in the iPMv and cPMv in the hUTC treated monkeys compared to placebos [F(1,6) = 12.82, p = 0.0116]. Abbreviations: iPMv, ipsilesional ventral premotor cortex. cPMv, contralesional ventral premotor cortex. iPMd, ipsilesional dorsal premotor cortex. cPMd, contralesional dorsal premotor cortex.

Figure 5. Uniform expression of synaptophysin puncta in the premotor cortices

Representative micrograph of synaptophysin puncta in ipsilesional ventral premotor cortex are shown. Examples of a synaptophysin puncta are shown at the white arrows. The asterisks represent areas of background. Scale bar: 1 μm

Figure 6. Synaptophysin puncta in bilateral premotor cortices remain unchanged following hUTC treatment

Numbers of synaptophysin puncta in the dorsal and ventral premotor cortex were estimated using unbiased stereology and are represented here (in millions). There was no significant difference between groups in any region. Abbreviations: iPMv, ipsilesional ventral premotor cortex. cPMv, contralesional ventral premotor cortex. iPMd, ipsilesional dorsal premotor cortex. cPMd, contralesional dorsal premotor cortex.

Figure 7. BrdU positive cells in the subventricular zone and subgranular zone

A representative micrograph of the iSVZ is shown (A). At a higher magnification (B), an example of a BrdU positive cell is shown at the black arrow and an endothelial cell (excluded from the current study) is shown at the red arrow. A representative micrograph of the ipsilesional dentate gyrus is shown (C). At a higher magnification (D), examples of two BrdU positive cells in the iSGZ are shown at the black arrows.

Figure 8. BrdU positive cells in Subgranular Zone increase following hUTC treatment

The densities of BrdU positive cells in the subventricular zone and subgranular zone were estimated using unbiased stereology. There was no significant difference between groups in any region. However, there are significantly more BrdU positive cells in cSGZ compared to iSGZ. [F(1,5) = 18.63, p = 0.0076]. Abbreviations: iSVZ, ipsilesional subventricular zone. cSVZ, contralesional subventricular zone. iSGZ, ipsilesional subgranular zone. cSGZ, contralesional subgranular zone.

Figure 9. Increased c-Fos activation is correlated with improved recovery time

Recovery time is defined as days following cortical injury to return to asymptotic performance in a 120 day period. There is a significant negative correlation between recovery time and the level of c-Fos activation in the iPMv (A) and cPMv (B).

Figure 10. Increased c-Fos activation is correlated with improved grasp topography

The mean grasp assessment score is reported on a scale from 0 (no movement) to 8 (preoperative finger/thumb opposition). There is a significant positive correlation between the mean grasp assessment score and the level of c-Fos activation in the iPMv (A) and cPMv (B).