Subacute neural stem cell therapy for traumatic brain injury

Abstract

Introduction: Traumatic brain injury (TBI) frequently results in devastating and prolonged morbidity. Cellular therapy is a burgeoning field of experimental treatment that has shown promise in the management of many diseases, including TBI. Previous work suggests that certain stem and progenitor cell populations migrate to sites of inflammation and improve functional outcome in rodents after neural injury. Unfortunately, recent study has revealed potential limitations of acute and intravenous stem cell therapy. We studied subacute, direct intracerebral neural stem and progenitor cell (NSC) therapy for TBI.

Materials and methods: The NSCs were characterized by flow cytometry and placed (400,000 cells in 50 muL 1x phosphate-buffered saline) into and around the direct injury area, using stereotactic guidance, of female Sprague Dawley rats 1 wk after undergoing a controlled cortical impact injury. Immunohistochemistry was used to identify cells located in the brain at 48 h and 2 wk after administration. Motor function was assessed using the neurological severity score, foot fault, rotarod, and beam balance. Cognitive function was assessed using the Morris water maze learning paradigm. Repeated measures analysis of variance with post-hoc analysis were used to determine significance at P < 0.05.

Results: Immunohistochemistry analysis revealed that 1.4-1.9% of infused cells remained in the neural tissue at 48 h and 2 wk post placement. Nearly all cells were located along injection tracks at 48 h. At 2 wk some cell dispersion was apparent. Rotarod motor testing revealed significant increases in maximal speed among NSC-treated rats compared with saline controls at d 4 (36.4 versus 27.1 rpm, P < 0.05) and 5 (35.8 versus 28.9 rpm, P < 0.05). All other motor and cognitive evaluations were not significantly different compared to controls.

Conclusions: Placement of NSCs led to the cells incorporating and remaining in the tissues 2 wk after placement. Motor function tests revealed improvements in the ability to run on a rotating rod; however, other motor and cognitive functions were not significantly improved by NSC therapy. Further examination of a dose response and optimization of placement strategy may improve long-term cell survival and maximize functional recovery.

FIG. 1

Intracerebral placement of NSCs. The dashed circle represents the location of the craniotomy and the black dots are locations of NSC injection. At each injection site, 40,000 NSCs in 5 μL of saline vehicle were injected 2–3 mm below the cortex surface. (Color version of figure is available online.)

FIG. 2

Flowchart outlining the timing of the experiments.

FIG. 3

Flow cytometric immunophenotyping of rat neural stem cells (rNSC). The rNSCs were immunophenotyped for the following receptors/proteins: Nestin, GFAP, β-tubulin III, CNPase and myelin basic protein (MBP). Cells were found to be Nestin-positive and mildly positive for GFAP and CNPase.

FIG. 4

Immunohistochemistry of brain 48 h after CCI injury and rNSC placement. At 48 h after injury, numerous rNSCs are seen in the penumbral cortex. The nuclei are stained with 4′,6-diamidino-2-phenylindole, dilactate (blue), and the rNSCs are stained with Qtracker 655 (red). The insert shows an entire coronal section after Nissl staining, with a red box outlining an area similar to the magnified image. (Color version of figure is available online.)

FIG. 5

Rotarod motor testing results. Rats were tested using 3 trials per day at d 8–12 (d 1–5 after NSC placement). Velocity of the rod was started at 15 rpm and increased 3 rpm every 5 s. Maximum speed (y-axis) maintained prior to failure (fall or inability to stay on the top of the rod) was recorded. On d 4 and 5, rats that received rNSCs were able to remain on the rotarod at a significantly increased speed relative to rats who received vehicle injection.

FIG. 6

Neurological severity score (NSS) results. Rats were evaluated for global neurological function using the previously described NSS on d 8–12 (d 1–5 after NSC placement). No statistically significant differences were identified.

FIG. 7

Morris water maze results. Two wk (early, A) and 10 wk (late, B) after injury (1 wk and 9 wk after NSC placement), animals were tested in a hidden platform, learning a paradigm version of the Morris water maze task. Animals were tested using 4 trials per day, with latency to the hidden platform (y-axis) recorded after each trial. No statistically significant differences were identified.