Timothy Schallert

Animal models of neurological deficits: how relevant is the rat?

Animal models of neurological deficits are essential for the assessment of new therapeutic options. It has been suggested that rats are not as appropriate as primates for the symptomatic modelling of disease, but a large body of data argues against this view. Comparative analyses of movements in rats and primates show homology of many motor patterns across species. Advances have been made in identifying rat equivalents of akinesia, tremor, postural deficits and dyskinesia, which are relevant to Parkinsons disease. Rat models of hemiplegia, neglect and tactile extinction are useful in assessing the outcome of ischaemic or traumatic brain injury, and in monitoring the effects of therapeutic interventions. Studies in rodents that emphasize careful behavioural analysis should continue to be developed as effective and inexpensive models that complement studies in primates.

Transgenerational epigenetic imprints on mate preference

Environmental contamination by endocrine-disrupting chemicals (EDC) can have epigenetic effects (by DNA methylation) on the germ line and promote disease across subsequent generations. In natural populations, both sexes may encounter affected as well as unaffected individuals during the breeding season, and any diminution in attractiveness could compromise reproductive success. Here we examine mate preference in male and female rats whose progenitors had been treated with the antiandrogenic fungicide vinclozolin. This effect is sex-specific, and we demonstrate that females three generations removed from the exposure discriminate and prefer males who do not have a history of exposure, whereas similarly epigenetically imprinted males do not exhibit such a preference. The observations suggest that the consequences of EDCs are not just transgenerational but can be “transpopulational”, because in many mammalian species, males are the dispersing sex. This result indicates that epigenetic transgenerational inheritance of EDC action represents an unappreciated force in sexual selection. Our observations provide direct experimental evidence for a role of epigenetics as a determinant factor in evolution.

Behavioral Tests After Intracerebral Hemorrhage in the Rat

Background and Purpose— In humans, intracerebral hemorrhage (ICH) causes marked perihematomal edema formation and neurological deficits. A rat ICH model, involving infusion of autologous blood into the caudate, has been used extensively to study mechanisms of edema formation, but an examination of behavioral outcome would improve its preclinical utility and provide a more rigorous assessment of the pathological cascade of events over time. The purpose of this study was to use a battery of sensorimotor function tests to examine the neurological effects of ICH in the rat and to examine which components of the hematoma are involved in generating those effects. Methods— The behavioral tests used were forelimb placing, preference for forelimb use for weight shifts during vertical exploration of a cylindrical enclosure, and a corner turn test. Rats were tested from day 1 to day 28 after injection of autologous whole blood; injection of blood plus hirudin (thrombin inhibitor), packed red blood cells, thrombin, or saline; or needle placement only. Results— The battery of tests indicated that there were marked neurological deficits by day 1 after ICH, with progressive recovery of function over 4 weeks. The forelimb placing score paralleled changes in edema. Injection of thrombin caused and injection of hirudin reduced the ICH-induced neurological deficits. Injection of packed red blood cells, which causes delayed edema formation, induced delayed neurological deficits Conclusions— These tests allow continuous monitoring of neurological deficits after rat ICH and assessment of therapeutic interventions. The time course of the neurological deficit closely matched the time course of cerebral edema for both ICH and injection of blood components. There was marked recovery of function after ICH, which may be amenable to therapeutic manipulation.

Overgrowth and pruning of dendrites in adult rats recovering from neocortical damage

Unilateral lesions to the forelimb representation (FL) area of the rat sensorimotor cortex caused a time-dependent increase in the dendritic arborization of layer V pyramidal neurons in the contralateral homotopic cortex. The increase in arborization was maximum at 2-3 weeks after the lesion, following which there was a partial reduction in dendritic branching. These neural morphological changes may be related to post-lesion behavioral changes in the use of the forelimbs.

Tactile extinction: distinguishing between sensorimotor and motor asymmetries in rats with unilateral nigrostriatal damage.

Abstract We describe and demonstrate the usefulness of a reliable procedure for quantifying “sensorimotor” asymmetries in rats with nigrostriatal damage. Conventional tests, such as the popular Von Frey hair technique are sometimes inadequate in that positive responses are difficult to quantify, they are susceptible to subjective interpretation, and they require that the animal make head movements. Circling behavior has become established as a measure of asymmetrical nigrostriatal activity, but it does not measure stimulus-directed movement. One group of rats was given unilateral microinfusions of 6-OHDA into the nigrostriatal system which decreased levels of dopamine in the ipsilateral striatum and yieded an ipsilateral turning bias. Another group was given electrolytic lesions in the substantia nigra area, which yielded a severe contralateral circling bias. Small pieces of adhesive paper were applied to various parts of the limbs or snout and latencies to remove the stimuli were recorded. Because head and body movements were not required, assessment of stimulus-directed movement asymmetries could be quantified rapidly without the interference of stimulus-independent (“spontaneous”) postural and motor asymmetries. In the 6-OHDA-treated rats there was ipsilateral bias in stimulus-directed activity. Indeed, there was ipsilateral hyper-reactivity relative to sham controls. In rats with electrolytic nigra lesions, despite a strong contralateral “spontaneous” motor bias, there likewise was an ipsilateral bias in stimulus-directed movement, which could only be detected using the adhesive removal test. This similarity in stimulus-directed movement asymmetry between the two groups may reflect a common asymmetry in ascending dopaminergic pathways. The opposite circling bias following electrolytic nigra lesions may reflect differential damage to non-dopaminergic efferent projections. The bilateral adhesive removal (tactile extinction) test appears to permit the separate quantification of stimulus-directed and stimulus-independent movement asymmetries.

Motor Enrichment and the Induction of Plasticity before or after Brain Injury

Voluntary exercise, treadmill activity, skills training, and forced limb use have been utilized in animal studies to promote brain plasticity and functional change. Motor enrichment may prime the brain to respond more adaptively to injury, in part by upregulating trophic factors such as GDNF, FGF-2, or BDNF. Discontinuation of exercise in advance of brain injury may cause levels of trophic factor expression to plummet below baseline, which may leave the brain more vulnerable to degeneration. Underfeeding and motor enrichment induce remarkably similar molecular and cellular changes that could underlie their beneficial effects in the aged or injured brain. Exercise begun before focal ischemic injury increases BDNF and other defenses against cell death and can maintain or expand motor representations defined by cortical microstimulation. Interfering with BDNF synthesis causes the motor representations to recede or disappear. Injury to the brain, even in sedentary rats, causes a small, gradual increase in astrocytic expression of neurotrophic factors in both local and remote brain regions. The neurotrophic factors may inoculate those areas against further damage and enable brain repair and use-dependent synaptogenesis associated with recovery of function or compensatory motor learning. Plasticity mechanisms are particularly active during time-windows early after focal cortical damage or exposure to dopamine neurotoxins. Motor and cognitive impairments may contribute to self-imposed behavioral impoverishment, leading to a reduced plasticity. For slow degenerative models, early forced forelimb use or exercise has been shown to halt cell loss, whereas delayed rehabilitation training is ineffective and disuse is prodegenerative. However, it is possible that, in the chronic stages after brain injury, a regimen of exercise would reactivate mechanisms of plasticity and thus enhance rehabilitation targeting residual functional deficits.

A test for detecting long-term sensorimotor dysfunction in the mouse after focal cerebral ischemia

The mouse is an excellent model for investigations of stroke and neural injury. However, there is a paucity of long term functional outcome measurements for the mouse. We, therefore, developed a sensorimotor functional test (corner test) and applied this test to a model of focal cerebral ischemia in the mouse. Male C57/6J mice (n=20) were subjected to embolic middle cerebral artery (MCA) occlusion. Reduction of cerebral blood flow (CBF) was measured by perfusion weighted MRI at 1 h after ischemia. The corner test, which is sensitive to chronic sensorimotor and postural symmetries, a general neurological test battery, and a foot fault test were performed between 2 and 90 days after ischemia. Infarct volume was measured at 90 days after ischemia. Multivariable analysis revealed that the corner test was highly predictive for infarct volume measured at 90 days after stroke, with R(2) values ranging from 0.73 to 0.93. The foot-fault test and neurological score did not detect chronic behavioral impairments. A significant (P<0.001) correlation between the infarct volume and the corner test was detected at 90 days after mild focal cerebral ischemia, whereas, there was no correlation between the infarct volume and neurological score or foot-fault. The data demonstrate that the corner test is a sensitive and objective test, which can be applied to evaluate long term functional outcome after stroke in the mouse.

Recovery of function after brain damage: Severe and chronic disruption by diazepam

Following unilateral damage to the anterior-medial region of the neocortex (AMC) in rats a sensory asymmetry appeared, but recovered within a week. In a separate group of rats with AMC lesions daily 3-week exposure to diazepam (Valium, 5 mg/kg) beginning 12 h after surgery caused recovery to be delayed indefinitely. The efficiency and speed (as opposed to symmetry) of behavior was not impaired. More than 9 weeks after discontinuation of diazepam (12 weeks postsurgery), recovery was still not apparent. Postmortem analysis ruled out lesion size as a contributing factor. In a second experiment undrugged animals with AMC lesions were allowed to recover for at least 3 weeks before being exposed to diazepam. These animals showed only a transient (2-day) reinstatement of asymmetry despite continuous drug treatment. We conclude that important mechanisms serving recovery of function may be vulnerable during a short period soon after brain damage.

Neuroprotective effects of prior limb use in 6-hydroxydopamine-treated rats: possible role of GDNF

Unilateral administration of 6‐hydroxydopamine (6‐OHDA) into the medial forebrain bundle (MFB) causes a loss of dopamine (DA) in the ipsilateral striatum and contralateral motor deficits. However, if a cast is placed on the ipsilateral limb during the first 7 days following 6‐OHDA infusion, forcing the animal to use its contralateral limb, both the behavioral and neurochemical deficits are reduced. Here, we examine the effect of forced reliance on a forelimb during the 7 days prior to ipsilateral infusion of 6‐OHDA on the deficits characteristic of this lesion model. Casted animals displayed no behavioral asymmetries as measured 14–28 days postlesion and a marked attenuation in the loss of striatal DA and its metabolites at 30 days. In addition, animals receiving a unilateral cast alone had an increase in glial cell‐line derived neurotrophic factor (GDNF) protein in the striatum corresponding to the overused limb. GDNF increased within 1 day after the onset of casting, peaked at 3 days, and returned to baseline within 7 days. These results suggest that preinjury forced limb‐use can prevent the behavioral and neurochemical deficits to the subsequent administration of 6‐OHDA and that this may be due in part to neuroprotective effects of GDNF.

Use-dependent exacerbation of brain damage occurs during an early post-lesion vulnerable period.

For a period of time after unilateral brain injury, surviving neural tissue surrounding the lesion may be vulnerable to extremely high behavioral demand. Previously, we found that lesions of the forelimb representation area of the sensorimotor cortex (FL-SMC) in rats increase in size substantially when the intact forelimb is immobilized with a plaster of paris cast during the first 15 days after surgery, which forces overuse of the impaired forelimb. The present study was designed to determine whether the adult brain is more vulnerable to forced overuse of the impaired forelimb during the first 7 days post-lesion than during the second 7 days post-lesion. Using behavioral tests of forelimb use and stereological analysis of remaining tissue volume 40 days after FL-SMC lesions, we found that forced overuse of the impaired forelimb during the first 7 days after the initial damage caused expansion of neural injury and greatly interfered with restoration of function. In contrast, forced overuse of the impaired forelimb during the second 7 days had no significant effect on lesion size but nevertheless interfered with restoration of function. Thus, surviving neural tissue in the damaged hemisphere and recovery of function appear to be vulnerable to prolonged forced overuse of the impaired forelimb throughout the first 15 days, but tissue loss was detectable only when the animal was forced to use the impaired forelimb during the first 7 days after injury.