Michael R. Hoane

Incomplete nigrostriatal dopaminergic cell loss and partial reductions in striatal dopamine produce akinesia, rigidity, tremor and cognitive deficits in middle-aged rats.

The present study was conducted to determine if the full array of parkinsonian symptoms could be detected in rats with nigrostriatal cell loss and striatal dopamine depletions similar to levels reported in the clinical setting, and to determine if older rats exhibit more robust parkinsonian deficits than younger rats. Young (2 months old) and middle-aged (12 months old) rats received bilateral striatal infusions of 6-OHDA, over the next 3 months they were assessed with a battery of behavioral tests, and then dopaminergic nigrostriatal cells and striatal dopamine and DOPAC levels were quantified. The results of the present study suggest that: (1) the full array of parkinsonian symptoms (i.e. akinesia, rigidity, tremor and visuospatial cognitive deficits) can be quantified in rats with incomplete nigrostriatal dopaminergic cell loss and partial reductions in striatal dopamine levels (2) parkinsonian symptoms were more evident in middle-aged rats with 6-OHDA infusions, and (3) there was evidence of substantial neuroplasticity in the older rats, but regardless of the age of the animal, endogenous compensatory mechanisms were unable to maintain striatal dopamine levels after rapid, lesion-induced nigrostriatal cell loss. These results suggest that using older rats with nigrostriatal dopaminergic cell loss and reductions in striatal dopamine levels similar to those in the clinical condition, and measuring behavioral deficits analogous to parkinsonian symptoms, might increase the predictive validity of pre-clinical rodent models.

Transplantation of neuronal and glial precursors dramatically improves sensorimotor function but not cognitive function in the traumatically injured brain.

Embryonic stem (ES) cells have been investigated in various animal models of neurodegenerative disease; however, few studies have examined the ability of ES cells to improve functional outcome following traumatic brain injury (TBI). The purpose of the present study was to examine the ability of pre-differentiated murine ES cells (neuronal and glial precursors) to improve functional outcome. Rats were prepared with a unilateral controlled cortical impact injury or sham and then transplanted 7 days later with 100K ES cells (WW6G) (~30% neurons) or media. Two days following transplantation rats were tested on a battery of behavioral tests. It was found that transplantation of ES cells improved behavioral outcome by reducing the initial magnitude of the deficit on the bilateral tactile removal and locomotor placing tests. ES cells also induced almost complete recovery on the vibrissae --> forelimb placing test, whereas, media-transplanted rats failed to show recovery. Acquisition of a reference memory task in the Morris water maze was not improved by transplantation of ES cells. Histological analysis revealed a large number of surviving ES cells in the lesion cavity and showed migration of ES cells into subcortical structures. It was found that transplantation of ES cells prevented the occurrence of multiple small necrotic cavities that were seen in the cortex adjacent to the lesion cavity in media transplanted rats. Additionally, ES cells transplants also significantly reduced lesion size. Results of this study suggest that ES cells that have been pre-differentiated into neuronal precursors prior to transplantation have therapeutic potential.

The effects of nicotinamide on apoptosis and blood–brain barrier breakdown following traumatic brain injury

Nicotinamide has been shown to protect against many of the pathophysiological factors associated with both ischemic and traumatic brain injuries. The present study evaluated the neuroprotective effect of nicotinamide on the breakdown of the blood-brain barrier (BBB) and apoptosis expression following traumatic brain injury (TBI). Animals were prepared with a unilateral cortical contusion injury (CCI). Fifteen minutes following injury the animals received either nicotinamide (500 mg/kg, ip) or 0.9% saline. The animals were perfused at 5, 24, and 72 h post-injury. BBB integrity was assessed by endogenous rat IgG immunoreactivity. Recent studies have shown that IgG immunoreactivity is a reliable measure of BBB integrity. The results indicated that IgG immunoreactivity was greatest at 5 h and declined at 24 h after injury. Nicotinamide significantly reduced IgG expression at every time point following injury. Apoptosis was examined using the TUNEL method. The results indicated that TUNEL immunoreactivity peaked at 24 h. TUNEL(+) cells were classified morphologically as nonapoptotic (Type I) or apoptotic (Type II) to verify that the neuroprotective effects of nicotinamide occur by inhibiting apoptosis or necrosis. Administration of nicotinamide significantly reduced the expression of all TUNEL(+) cells in the tissue surrounding the lesion cavity. Specifically there was a significant reduction in the number of Type I, Type II, and Total TUNEL(+) cells in the nicotinamide-treated animals. In addition, nicotinamide reduced lesion cavity expansion 72 h following CCI. These findings suggest that nicotinamide reduces BBB breach and neuronal cell loss acutely following injury and that these reductions may account for the beneficial behavioral effects seen in previous studies.

Treatment with vitamin B3 improves functional recovery and reduces GFAP expression following traumatic brain injury in rats.

Previous studies have shown that administration of vitamin B(3) (B(3)) in animal models of ischemia significantly reduced the size of infarction and improved functional recovery. The present study evaluated the effect of administration of B(3) on recovery of function following traumatic brain injury (TBI), incorporating the bilateral medial frontal cortex contusion injury model. Groups of rats were assigned to B(3) (500 mg/kg) or saline (1.0 ml/kg) treatment conditions and received contusion injuries or sham surgeries. Drug treatment was administered 15 min and 24 h following injury. Rats were examined on a variety of tests to measure sensorimotor performance (bilateral tactile adhesive removal), skilled forelimb use (staircase test), and cognitive ability (reference and working memory) in the Morris Water Maze. Administration of B(3) following injury significantly reduced the behavioral impairments observed on the bilateral tactile removal test, but not on skilled forelimb use. The acquisition of reference and working memory tests were also significantly improved compared to saline-treated rats. Examination of the brains revealed that administration of B(3) significantly reduced the size of the lesion compared to treatment with saline. In addition, examination of glial fibrillary acidic protein (GFAP) expression around the lesion revealed that B(3) significantly reduced the number of GFAP(+) astrocytes. These results indicate that B(3) administration significantly improved behavioral outcome following injury, reduced the size of the lesion, and reduced the expression of GFAP. The current findings suggest that B(3) may have therapeutic potential for the treatment of TBI.

Nicotinamide reduces acute cortical neuronal death and edema in the traumatically injured brain

Previous studies have shown that administration of nicotinamide (Vitamin B(3)) in animal models of traumatic brain injury (TBI) and ischemia significantly reduced the size of infarction or injury and improved functional recovery. The present study evaluated the ability of nicotinamide to provide acute neuroprotection and edema reduction following TBI. Groups of rats were assigned to nicotinamide (500mg/kg) or saline (1.0ml/kg) treatment conditions and received contusion injuries or sham surgeries. Drug treatment was administered 15min following injury. Brains were harvested 24h later and either processed for histology or water content. Frozen sections were stained with the degenerating neuron stain (Fluoro-Jade B) (FJ) and cell counts were performed at the site of injury. Additional brains were processed for water content (a measure of injury-induced edema). Results of this study showed that administration of nicotinamide following TBI significantly reduced the number of FJ(+) neurons in the injured cortex compared to saline-treated animals. Examination of the water content of the brains also revealed that administration of nicotinamide significantly attenuated the amount of water compared to saline-treated animals in the injured cortex. These results indicate that nicotinamide administration significantly reduced neuronal death and attenuated cerebral edema following injury. The current findings suggest that nicotinamide significantly modulates acute pathophysiological processes following injury and that this may account for its beneficial effects on recovery of function following injury.

Nicotinamide treatment induces behavioral recovery when administered up to 4 hours following cortical contusion injury in the rat

Recent studies have demonstrated nicotinamide (NAM), a soluble B-group vitamin, to be an effective treatment in experimental models of traumatic brain injury (TBI). However, research on this compound has been limited to administration regimens starting shortly after injury. This study was conducted to establish the window of opportunity for NAM administration following controlled cortical impact (CCI) injury to the frontal cortex. Groups of rats were assigned to NAM (50 mg/kg), saline (1 ml/kg), or sham conditions and received contusion injuries or sham procedures. Injections of NAM or saline were administered at 15 min, 4 h, or 8 h post-injury, followed by five boosters at 24 h intervals. Following the last injection, blood was taken for serum NAM analysis. Animals were tested on a variety of tasks to assess somatosensory performance (bilateral tactile adhesive removal and vibrissae-forelimb placement) and cognitive performance (reference and working memory) in the Morris water maze. The results of the serum NAM analysis showed that NAM levels were significantly elevated in treated animals. Behavioral analysis on the tactile removal test showed that all NAM-treated groups facilitated recovery of function compared with saline treatment. On the vibrissae-forelimb placing test all NAM-treated groups also were significantly different from the saline-treated group. However, the acquisition of reference memory was only significantly improved in the 15-min and 4-h groups. In the working memory task both the 15-min and 4-h groups also improved working memory compared with saline treatment. The window of opportunity for NAM treatment is task-dependent and extends to 8 h for the sensorimotor tests but only extends to 4 h post-injury in the cognitive tests. These results suggest that a 50 mg/kg treatment regimen starting at the clinically relevant time point of 4 h may result in attenuated injury severity in the human TBI population.

Nicotinamide treatment provides acute neuroprotection and GFAP regulation following fluid percussion injury.

Previous studies in our laboratory have demonstrated the preclinical efficacy of nicotinamide (NAM; vitamin B3) treatment following fluid percussion injury (FPI). At a dose of 500 or 50 mg/kg, NAM significantly facilitated recovery of function on a variety of motor and sensorimotor tasks in rodents, and the 500 mg/kg dose improved cognitive performance. The purpose of the present study was to examine the acute neuroprotective ability of NAM following FPI. Rats were given a moderate FPI (1.8 atm) or sham injury. NAM (500 or 50 mg/kg) or saline was administered 15 min and 20 h after FPI. Rats were sacrificed at 24 h or 7 days following injury and prepared for histological analysis. Systematic volumetric measurements were conducted to examine cortical loss in a series of cresyl violet stained slices to examine the development of the injury cavity. To assess the extent of astrocytic activity and neurodegeneration, triple labeling with glial fibrillary acidic protein (GFAP), Fluoro-Jade B (FJ), and DAPI was performed. GFAP(+) astrocytes and FJ(+) neurons in the ipsilateral and contralateral cortex, and ipsilateral hippocampus and thalamus were assessed. While not significant at 24 h, NAM significantly attenuated cortical tissue loss at 7 days. At 24 h, the number of GFAP(+) astrocytes was significantly reduced by NAM. However, the inverse was observed at 7 days where NAM treatment significantly increased the number of GFAP(+) astrocytes. Both doses of NAM also significantly reduced FJ expression at the 24-h and 7-day time intervals. The results of this study suggest that NAM has strong neuroprotective abilities in the injured brain and may have therapeutic potential for brain injury.

Administration of raloxifene reduces sensorimotor and working memory deficits following traumatic brain injury.

Hormonal differences between males and females have surfaced as a crucial component in the search for effective treatments after experimental models of traumatic brain injury (TBI). Recent findings have shown that selective estrogen receptor modulators (SERMs) may have therapeutic benefit. The present study examined the effects of raloxifene, a SERM, on functional recovery after bilateral cortical contusion injury (bCCI) or sham procedure. Male rats received injections of raloxifene (3.0mg/kg, i.p.) or vehicle (1.0 ml/kg, i.p.) 15 min, 24, 48, 72, and 96 h after bCCI or sham procedure. Rats were tested on both sensorimotor (bilateral tactile removal and locomotor placing tests) and cognitive tests (reference and working memory in the Morris water maze). Raloxifene-treated animals showed a significant reduction in the initial magnitude of the deficit and facilitated the rate of recovery for the bilateral tactile removal test, compared to vehicle-treated animals. The raloxifene-treated animals also showed a significant improvement in the acquisition of working memory compared to vehicle-treated animals. However, raloxifene did not significantly improve the acquisition of reference memory or locomotor placing ability. Raloxifene treatment also did not result in a significant reduction in the size of the lesion cavity. Thus, the task-dependent improvements seen following raloxifene treatment do not appear to be the result of cortical neuroprotection. However, these results suggest that raloxifene improves functional outcome following bCCI and may present an interesting avenue for future research.

Transplantation of GABAergic neurons but not astrocytes induces recovery of sensorimotor function in the traumatically injured brain.

Embryonic stem (ES) cells have been investigated in many animal models of injury and disease. However, few studies have examined the ability of pre-differentiated ES cells to improve functional outcome following traumatic brain injury (TBI). The purpose of the present study was to compare the effect of murine ES cells that were pre-differentiated into GABAergic neurons or astrocytes on functional recovery following TBI. Neural and astrocyte induction was achieved by co-culturing ES cells on a bone marrow stromal fibroblast (M2-10B4) feeder layer and incubating them with various mitogenic factors. Rats were initially prepared with a unilateral controlled cortical contusion injury of the sensorimotor cortex or sham procedure. Rats were transplanted 7 days following injury with approximately 100K GABAergic neurons, astrocytes, fibroblasts, or media. Animals were assessed on a battery of sensorimotor tasks following transplantation. The stromal fibroblast cells (M2-10B4), as a control cell line, did not differ significantly from media infusions. Transplantation of GABAergic neurons facilitated complete and total recovery on the vibrissae-forelimb placing test as opposed to all other groups, which failed to show any recovery. It was also found that GABAergic neurons reduced the magnitude of the initial impairment on the limb use test. Histological analysis revealed infiltration of host brain with transplanted neurons and astrocytes. The results of the present study suggest that transplantation of pre-differentiated GABAergic neurons significantly induces recovery of sensorimotor function; whereas, astrocytes do not.

COG1410 Improves Cognitive Performance and Reduces Cortical Neuronal Loss in the Traumatically Injured Brain

We have previously shown that a single dose of COG1410, a small molecule ApoE-mimetic peptide derived from the apolipoprotein E (ApoE) receptor binding region, improves sensorimotor and motor outcome following cortical contusion injury (CCI). The present study evaluated a regimen of COG1410 following frontal CCI in order to examine its preclinical efficacy on cognitive recovery. Animals were prepared with a bilateral CCI of the frontal cortex. A regimen of COG1410 (0.8mg/kg intravenously [IV]) was administered twice, at 30min and again at 24h post-CCI. Starting on day 11, the animals were tested for their acquisition of a reference memory task in the Morris water maze (MWM), followed by a working memory task in the MWM on day 15. Following CCI, the animals were also tested on the bilateral tactile adhesive removal test to measure sensorimotor dysfunction. On all of the behavioral tests the COG1410 group was no different from the uninjured sham group. Administration of the regimen of COG1410 significantly improved recovery on the reference and working memory tests, as well as on the sensorimotor test. Lesion analysis revealed that COG1410 significantly reduced the size of the injury cavity. Administration of COG1410 also reduced the number of degenerating neurons, as measured by Fluoro-Jade C staining, in the frontal cortex at 48h post-CCI. These results suggest that a regimen of COG1410 appeared to block the development of significant behavioral deficits and reduced tissue loss. These combined findings suggest that COG1410 appears to have strong preclinical efficacy when administered following traumatic brain injury (TBI).