R. Andrew Tasker

Effectiveness of PSD95 Inhibitors in Permanent and Transient Focal Ischemia in the Rat

Background and Purpose— Postsynaptic density-95 inhibitors reduce ischemic brain damage without inhibiting excitatory neurotransmission, circumventing the negative consequences of glutamatergic inhibition. However, their efficacy in permanent ischemia and in providing permanent neuroprotection and neurobehavioral improvement in a practical therapeutic window is unproven. These were tested here under conditions that included fever, which is a common occurrence in clinical stroke. Methods— Six studies were performed in unfasted Sprague-Dawley rats. Two involved permanent pial vessel occlusion in male and female rats. Two involved permanent middle cerebral artery occlusion, which induced severe hyperthermia, and 2 involved transient middle cerebral artery occlusion. Animals were treated with a single intravenous injection of postsynaptic density-95 inhibitors (Tat-NR2B9c[SDV] or Tat-NR2B9c[TDV]) 1 hour or 3 hours after stroke. Infarct volumes and neurobehavior were assessed in a blinded manner at 24 hours (pial vessel occlusion and permanent middle cerebral artery occlusion) or at 62 days (transient middle cerebral artery occlusion). Results— Postsynaptic density-95 inhibitors dramatically reduced infarct size in male and female animals exposed to pial vessel occlusion (>50%), in hyperthermic animals with fever exceeding 39°C exposed to permanent middle cerebral artery occlusion (approximately 50%), and at 62 days poststroke in animals exposed to transient middle cerebral artery occlusion (approximately 80%). Effectiveness of postsynaptic density-95 inhibitors was achieved without the drugs affecting body temperature. In transient middle cerebral artery occlusion, a single dose of postsynaptic density-95 inhibitor given 3 hours after stroke onset permanently maintained reduced infarct size and improved neurobehavior. Conclusions— Postsynaptic density-95 inhibitors administrated 3 hours after stroke onset reduced infarct volumes and improved long-term neurobehavioral functions in a wide therapeutic window. This raises the possibility that they may have future clinical usefulness.

Low doses of non-NMDA glutamate receptor agonists alter neurobehavioural development in the rat

While it is known that glutamate is critical to CNS development and function, less is known about the role of kainate receptors, a subclass of ionotropic glutamate receptors, during ontogeny. This is especially true with respect to the emergence and expression of behaviour. It is also known that the neonatal CNS differs from that of adults with respect to excitatory amino acid (EAA) toxicity. Our aim was to determine the effects of early low-dose stimulation of kainate receptors on physical and behavioural development in the rat. Saline, one of two subtoxic doses of domoic acid (DOM) (5 and 20 microg/kg), or in a parallel study, saline, or one of two pharmacologically equivalent doses of kainic acid (KA) (25 and 100 microg/kg), were systemically administered once daily from postnatal days (PNDs) 8-14. While DOM or KA had no effect on typical measures of toxicity such as weight gain, acoustic startle, ultrasonic vocalizations (UVs), or maternal retrieval, these doses were shown to be physiologically relevant, producing particular group differences in eye opening, conditioned place preference, and activity levels. We conclude that administration of very low doses of selective kainate receptor agonists during the second postnatal week produces changes in neurobehavioural development in the rat.

Selective reductions in subpopulations of GABAergic neurons in a developmental rat model of epilepsy

In the rat, early postnatal development is a critical period for neuronal migration, differentiation and network formation, requiring appropriate and timely glutamate and gamma-aminobutyric acid (GABA) signaling. Insults that affect either of these systems may result in increased excitatory activity, potentially leading to changes in neuronal proliferation and/or connectivity. We have previously shown that postnatal administration of low doses of domoic acid (DOM) can produce many of the behavioral and morphological changes found in current animal models of temporal lobe epilepsy (TLE), as well as the human condition. Using immunohistochemical techniques, we sought to characterize alterations in specific hippocampal GABAergic subpopulations at various locations along the septo-temporal axis in the DOM model. Results show decreased levels of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) in the ventral hilus and region- and sex-specific reductions in parvalbumin (PV)-containing immunoreactivity, but no alterations in somatostatin (SST) expression. These regional and sex-dependent changes in specific subpopulations of GABAergic interneurons may contribute to seizure development in this slowly progressing developmental model of TLE, and highlight how even subtle intervention may alter the interplay between glutamate and GABA systems during critical developmental stages.

Perinatal asphyxia: current status and approaches towards neuroprotective strategies, with focus on sentinel proteins

Delivery is a stressful and risky event menacing the newborn. The mother-dependent respiration has to be replaced by autonomous pulmonary breathing immediately after delivery. If delayed, it may lead to deficient oxygen supply compromising survival and development of the central nervous system. Lack of oxygen availability gives rise to depletion of NAD+ tissue stores, decrease of ATP formation, weakening of the electron transport pump and anaerobic metabolism and acidosis, leading necessarily to death if oxygenation is not promptly re-established. Re-oxygenation triggers a cascade of compensatory biochemical events to restore function, which may be accompanied by improper homeostasis and oxidative stress. Consequences may be incomplete recovery, or excess reactions that worsen the biological outcome by disturbed metabolism and/or imbalance produced by over-expression of alternative metabolic pathways. Perinatal asphyxia has been associated with severe neurological and psychiatric sequelae with delayed clinical onset. No specific treatments have yet been established. In the clinical setting, after resuscitation of an infant with birth asphyxia, the emphasis is on supportive therapy. Several interventions have been proposed to attenuate secondary neuronal injuries elicited by asphyxia, including hypothermia. Although promising, the clinical efficacy of hypothermia has not been fully demonstrated. It is evident that new approaches are warranted. The purpose of this review is to discuss the concept of sentinel proteins as targets for neuroprotection. Several sentinel proteins have been described to protect the integrity of the genome (e.g. PARP-1; XRCC1; DNA ligase IIIα; DNA polymerase β, ERCC2, DNA-dependent protein kinases). They act by eliciting metabolic cascades leading to (i) activation of cell survival and neurotrophic pathways; (ii) early and delayed programmed cell death, and (iii) promotion of cell proliferation, differentiation, neuritogenesis and synaptogenesis. It is proposed that sentinel proteins can be used as markers for characterising long-term effects of perinatal asphyxia, and as targets for novel therapeutic development and innovative strategies for neonatal care.

Transient domoic acid excitotoxicity increases BDNF expression and activates both MEK- and PKA-dependent neurogenesis in organotypic hippocampal slices

BackgroundWe have previously reported evidence of cell proliferation and increased neurogenesis in rat organotypic hippocampal slice cultures (OHSC) after a transient excitotoxic injury to the hippocampal CA1 area induced by low concentrations of the AMPA/kainate agonist domoic acid (DOM). An increased baseline rate of neurogenesis may contribute to recovery from DOM-induced mild injury but the intracellular mechanism(s) responsible for neuronal proliferation remain unclear. The current study investigated the key intracellular pathways responsible for DOM-induced neurogenesis in OHSC including the effects of transient excitotoxicity on the expression of brain-derived neurotrophic factor (BDNF), a well-known regulator of progenitor cell mitosis.ResultsApplication of a low concentration of DOM (2 μM) for 24 h followed by recovery induced a significant and long lasting increase in BDNF protein levels expressed by both neurons and microglial cells. Furthermore, the mild DOM toxicity stimulated both PKA and MEK-dependent intracellular signaling cascades and induced a significant increase in BDNF- transcription factor CREB activation and BDNF-receptor TrkB expression. Coexposure to specific inhibitors of PKA and MEK phosphorylation resulted in a significant decrease in the neurogenic marker doublecortin.ConclusionsOur results suggest that transient excitotoxic insult induced by DOM produces BDNF and CREB overexpression via MEK and PKA pathways and that both pathways mediate, at least in part, the increased neural proliferation resulting from mild excitotoxicity.

Low-dose neonatal domoic acid causes persistent changes in behavioural and molecular indicators of stress response in rats

Appropriate stress responses rely on a finely-tuned neuronal balance that must continually adapt to a frequently changing external environment. Alterations in this balance can result in susceptibility to a variety of stress-related disorders, as well as exacerbate already existing conditions. We have previously reported that rat pups injected with a very low dose (20 μg/kg) of domoic acid during the second postnatal week of life display low-grade seizure behaviours when challenged with stressful tasks, and also exhibit a variety of structural and functional changes similar to those seen in temporal lobe epilepsy. The current study was designed to investigate markers of altered stress-response in this model. Following neonatal treatment, adult rats were tested in the elevated plus maze, as well as two water maze tasks, both of which involved a platform reversal challenge. Results indicated a modified behavioural stress/anxiety response, increased perseveration, and alterations in search strategy for all domoate-treated rats, as well as male-specific deficits in cognitive flexibility. In addition, 80% of treated males and 20% of treated females exhibited seizure behaviour. Western blot analysis revealed male-only increases in adrenergic receptor (α2a and α2c) and mineralocorticoid receptor expression, and subtle sex-specific changes in glucocorticoid receptor expression, but no differences in corticotropin-releasing factor receptors I/II, or dopamine D2 receptor expression. A significant decrease in glucocorticoid:mineralocorticoid ratio was also noted. We conclude that early exposure to DOM alters central mechanisms underlying stress response, and that this model may be valuable for investigating the connection between stress and neurological disorders.

An improved post-operative care protocol allows detection of long-term functional deficits following MCAo surgery in rats

Developing new therapeutants for stroke requires animal models in which typical stroke outcomes can be detected. In rats, temporary occlusion of the middle cerebral artery (MCAo) closely resembles reversible human ischemic stroke, but most neuroprotection studies have used limited, short-term (1-2 weeks) behavioural and histological endpoints in this model. Further, the use of this model for assessing long-term recovery has been questioned. Long-term deficits may be difficult to detect because testing may not reflect the diversity of functional outcomes in clinical stroke, and/or high mortality rates mean that only the least severely affected rats remain to be tested. We believe that enhanced survival through intensive post-operative husbandry practices, and extensive behavioural testing that reflects multiple clinically relevant behavioural endpoints, will permit the detection of long-term functional deficits. In the present study, male SD rats (280-320 g) received transient (90 min) MCAo (n = 19) or sham surgery (n = 13). An intense post-operative care protocol was maintained, and assessments included various physical and sensorimotor parameters, tests of emotionality and tests of learning and memory. We report 0% mortality, and statistically significant deficits on all aspects of this battery, including learning and memory deficits up to 2 months post-MCAo. The current study demonstrates that with adequate post-operative care and extensive behavioural testing, assessing the potential of new therapeutants for promoting long-term functional neuroprotection following MCAo in the rat is feasible.

Voluntary forced use of the impaired limb following stroke facilitates functional recovery in the rat

Constraint induced movement therapy (CIMT), which forces use of the impaired arm following stroke, improves functional recovery. The mechanisms underlying recovery are not well understood, necessitating further investigation into how rehabilitation may affect neuroplasticity using animal models. Animal motivation and stress make modelling CIMT in animals challenging. We have shown that following focal ischemia, voluntary forced use therapy using pet activity balls could engage the impaired forelimb and result in a modest acceleration in recovery. In this study, we investigated the effects of a more intensive appetitively motivated regimen that included task specific reaching exercises. Adult male Sprague Dawley rats were subjected to focal unilateral stroke using intracerebral injections of endothelin-1 or sham surgery. Three days later, stroke animals were assigned to daily rehabilitation or control therapy. Rehabilitation consisted of 30 min of generalized movement sessions in activity balls, followed by 30 min of voluntary task-specific movement using reaching boxes. Rats were tested weekly to measure forelimb deficit and recovery. After 30 days, animals were euthanized and tissue was examined for infarct volume, brain derived neurotrophic factor expression, and the presence of new neurons using doublecortin immunohistochemistry. Rehabilitation resulted in a significant acceleration of forelimb recovery in several tests, and a significant increase in the number of doublecortin-expressing cells. Furthermore, while the proportion of cells expressing BDNF in the peri-infarct region did not change, there was a shift in the cellular origin of expressed BDNF, resulting in significantly more non-neuronal, non-astrocytic BDNF, presumed to be of microglial origin.

Use of osmotic minipumps for sustained drug delivery in rat pups: effects on physical and neurobehavioural development

Osmotic minipumps are often used as an alternative to repetitive injections for prolonged drug delivery in adult rats. The appropriateness of using this technology for sustained drug delivery in newborn rats, however, has not been validated. Our objective was to determine if implantation of osmotic minipumps, and the associated surgical stress, during a critical developmental period, affects early development and subsequent behaviour. SD rat pups were assigned to control, minipump, or sham surgery treatment conditions (n=12/group). On P8, pups were briefly anaesthetised with isoflurane in oxygen, and Alzet 1007D osmotic minipumps, loaded with normal saline, were aseptically implanted (removed on P17). Sham-treated rats received identical treatment (with the exception of pump placement), while control pups were left undisturbed. Development was examined daily using a standard test battery (P9-P21), and learning and memory in pups was assessed in a T-maze (P15, P17 and P19). Weight (P27 and P72), open-field (P25, P26 and P27) and novel water maze performance (P60-P72) were examined in the resulting adult. With the exception of a transient decrease in weight gain, pump-treated animals did not differ from either sham or control rats, on any pre- or postweaning assessment. Based on these results we conclude that the use of osmotic minipumps in rat pups is a viable alternative to repeated injections for sustained drug delivery.

Alterations to prepulse inhibition magnitude and latency in adult rats following neonatal treatment with domoic acid and social isolation rearing.

Deficits in perceptual, informational, and attentional processing are consistently identified as a core feature in schizophrenia and related neuropsychiatric disorders. Neonatal injections of low doses of the AMPA/kainate agonist domoic acid (DOM) have previously been shown to alter various aspects of perceptual and attentional processing in adult rats. The current study investigated the effects of combined neonatal DOM treatment with isolation rearing on prepulse inhibition behaviour and relevant neurochemical measures, to assess the usefulness of these paradigms in modeling neurodevelopmental disorders. Daily subcutaneous injections of DOM (20 μg/kg) or saline were administered to male and female rat pups from postnatal days (PND) 8-14. After weaning, rats were either housed alone or in groups of 4. Both the magnitude and latency of prepulse inhibition were determined in adulthood (approximately 4.5 months of age) and post-mortem brain tissue was assayed using Western blot. Social isolation alone significantly lowered PPI magnitude in male (but not female) rats while DOM treatment appeared to make animals refractory to this effect. Combining social isolation and DOM treatment caused an additive decrease in PPI startle latency. No statistically significant differences were found in the expression of D1, D2, TH, GAD65 or GAD67 protein in either the prefrontal cortex or hippocampus, although some tendencies toward differences were noted. We conclude that both neonatal low-dose DOM and social isolation affect prepulse inhibition in rats but that each paradigm exerts these effects through different neuronal signalling systems.