A heat-shock protein co-inducer treatment improves behavioral performance in rats exposed to hypoxia.

We investigated the effect of a heat-shock protein co-inducer, arimoclomol (CytRx, LA, CA), on hypoxia-adaptive responses using a rat model of simulated altitude exposure (hypobaric hypoxia).Cognitive function was measured using a T-maze and an object recognition test.Motor function was measured using an inclined-screen test and an adhesion removal test. Immunohistochemical analyses were assessed in brain for heat-shock protein 70 (HSP 70), intercellular adhesion molecule 1 (ICAM- 1) and apoptosis (TUNEL staining). Results show that both cognitive and motor performances were improved in rats treated with arimoclomol during hypoxic exposure; the hypoxia-induced expression of HSP70 and ICAM-1, and TUNEL-positive cells were reduced in brain with the treatment.Our data suggest that the arimoclomol treatment reduces the hypoxia-induced stress in brain tissue, and also improves the behavioral performance in rats during hypoxic adaptation.

Effects of progesterone on neurologic and morphologic outcome following diffuse traumatic brain injury in rats.

Previous studies have identified that progesterone may be neuroprotective following traumatic brain injury (TBI). However, most of these have utilized models of TBI that produce a focal lesion or a significant ischemic component, neither of which is necessarily present in diffuse TBI. The current study uses a model of diffuse TBI in rats to examine the effects of progesterone on morphological changes and functional outcome following TBI. Male and ovariectomized female rats were subject to severe impact-acceleration injury under halothane anesthesia. After injury, animals were given a physiological, subcutaneous dose of progesterone (1.67 mg/kg) or equal volume of vehicle (sesame oil) daily throughout a 9-day neurologic assessment period where functional outcome was assessed using the rotarod and Barnes maze tests. There was a similar post-injury performance of male and ovariectomized female animals. Post-injury administration of progesterone improved the motor and cognitive performance of ovariectomized and male animals compared to vehicle-treated controls. Morphological differences between these animals, such as dark cell change, caspase-3 and APP immunoreactivity, were also investigated. Progesterone-treated males showed comparatively less dead or dying neurons, and marked attenuation of caspase-3 immunoreactivity. Both ovariectomized female and male animals treated with progesterone showed a profound reduction in axonal injury (seen via diminished APP immunoreactivity) when compared to controls. We conclude that physiological concentrations of progesterone administered after diffuse TBI confers beneficial effects on morphologic and functional outcome in both ovariectomized female and male animals.

Both estrogen and progesterone attenuate edema formation following diffuse traumatic brain injury in rats.

Females have reduced brain edema compared to males after experimental brain trauma, although contradictory reports exist as to whether this is due to either estrogen or progesterone. In the present study, we demonstrate in both male and ovariectomized female rats that a single physiological dose of either hormone at 30 min after diffuse traumatic brain injury reduces both blood brain barrier permeability and edema formation. We conclude that both hormones may contribute to reduce edema in females after brain injury.

Role of the cell cycle in the pathobiology of central nervous system trauma.

Upregulation of cell cycle proteins occurs in both mitotic and post-mitotic neural cells after central nervous system (CNS) injury in adult animals. In mitotic cells, such as astroglia and microglia, they induce proliferation, whereas in post-mitotic cells such as neurons they initiate caspase-related apoptosis. We recently reported that early central administration of the cell cycle inhibitor flavopiridol after experimental traumatic brain injury (TBI) significantly reduced lesion volume, scar formation and neuronal cell death, while promoting near complete behavioral recovery. Here we show that in primary neuronal or astrocyte cultures structurally different cell cycle inhibitors (flavopiridol, roscovitine, and olomoucine) significantly reduce upregulation of cell cycle proteins, attenuate neuronal cell death induced by etoposide, and decrease astrocyte proliferation. Flavopiridol, in a concentration dependent manner, also attenuates proliferation/activation of microglia. In addition, we demonstrate that central administration of flavopiridol improves functional outcome in dose-dependent manner after fluid percussion induced brain injury in rats. Moreover, delayed systemic administration of flavopiridol significantly reduces brain lesion volume and edema development after TBI. These data provide further support for the therapeutic potential of cell cycle inhibitors for the treatment of clinical CNS injury and that protective mechanisms likely include reduction of neuronal cell death, inhibition of glial proliferation and attenuation of microglial activation.

Cell cycle inhibition provides neuroprotection and reduces glial proliferation and scar formation after traumatic brain injury.

Traumatic brain injury (TBI) causes neuronal apoptosis, inflammation, and reactive astrogliosis, which contribute to secondary tissue loss, impaired regeneration, and associated functional disabilities. Here, we show that up-regulation of cell cycle components is associated with caspase-mediated neuronal apoptosis and glial proliferation after TBI in rats. In primary neuronal and astrocyte cultures, cell cycle inhibition (including the cyclin-dependent kinase inhibitors flavopiridol, roscovitine, and olomoucine) reduced up-regulation of cell cycle proteins, limited neuronal cell death after etoposide-induced DNA damage, and attenuated astrocyte proliferation. After TBI in rats, flavopiridol reduced cyclin D1 expression in neurons and glia in ipsilateral cortex and hippocampus. Treatment also decreased neuronal cell death and lesion volume, reduced astroglial scar formation and microglial activation, and improved motor and cognitive recovery. The ability of cell cycle inhibition to decrease both neuronal cell death and reactive gliosis after experimental TBI suggests that this treatment approach may be useful clinically.

Neuroprotective effects of novel small peptides in vitro and after brain injury.

Thyrotropin-releasing hormone (TRH) and TRH analogues have been reported to be neuroprotective in experimental models of spinal cord injury and head injury. We have previously shown that a diketopiperazine structurally related to the TRH metabolite cyclo-his-pro reduces neuronal cell death in vitro and in vivo. Here we report the neuroprotective activity of other cyclic dipeptides in multiple in vitro models of neuronal injury and after controlled cortical impact (CCI) in mice. Using primary neuronal cultures, three novel dipeptides were compared to the previously reported diketopiperazine as well as to vehicle controls; each of the compounds reduced cell death after direct physical trauma or trophic withdrawal. Two of these peptides also protected against glutamate toxicity and beta-amyloid-induced injury; the latter also strongly inhibited glutamate-induced increases in intracellular calcium. Treatment with each of the test compounds resulted in highly significant improvement of motor and cognitive recovery after CCI, as well as markedly reducing lesion volumes as shown by high field magnetic resonance imaging. DNA microarray studies following fluid percussion induced traumatic brain injury (TBI) in rats showed that treatment with one of these dipeptides after injury significantly down-regulated expression of mRNAs for cell cycle proteins, aquaporins, cathepsins and calpain in ipsilateral cortex and/or hippocampus, while up-regulating expression of brain-derived neurotrophic factor, hypoxia-inducible factor and several heat-shock proteins. Many of these mRNA expression changes were paralleled at the protein level. The fact that these small peptides modulate multiple mechanisms favoring neuronal cell survival, as well as their ability to improve functional outcome and reduce posttraumatic lesion size, suggests that they may have potential utility in clinical head injury.

Caspase inhibitor z-DEVD-fmk attenuates calpain and necrotic cell death in vitro and after traumatic brain injury.

In studies designed to evaluate the therapeutic window for treatment of traumatic brain injury, the caspase 3 inhibitor z-DEVD-fmk improved neurologic function and reduced lesion volumes when administered at 1 but not at 4, 8, or 24 hours after injury. Moreover, neither caspase 3 nor PARP, a caspase 3 substrate, were cleaved in injured, untreated cortex from 1 to 72 hours after injury. Few cortical neurons expressed active caspase 3 or were TUNEL positive from 6 to 24 hours after injury, and TUNEL staining was primarily Type I (necrotic). Nissl staining revealed extensive neuronal necrosis in the injured cortex from 6 to 24 hours after impact. Considered together, these data suggested that z-DEVD-fmk may reduce neuronal necrosis, so we used an in vitro model of necrotic cell death induced by maitotoxin to test this further and explore the potential mechanism(s) involved. Z-DEVD-fmk (1 nM-100 microM) significantly attenuated maitotoxin induced neuronal cell death and markedly reduced expression of the 145 kD calpain-mediated alpha-spectrin breakdown product after maitotoxin injury. Neither the 120 kD caspase-mediated alpha-spectrin cleavage product nor cathepsin B were expressed after maitotoxin injury. In a cell free assay, z-DEVD-fmk reduced hydrolysis of casein by purified calpain I. Finally, z-DEVD-fmk reduced expression of the 145 kD calpain-mediated alpha-spectrin cleavage fragment after traumatic brain injury in vivo. These data suggest that neuroprotection by z-DEVD-fmk may, in part, reflect inhibition of calpain-related necrotic cell death.

Novel small peptides with neuroprotective and nootropic properties.

The tripeptide thyrotropin-releasing hormone (TRH) and/or related analogues have shown neuroprotective activity across multiple animal trauma models as well as in a small clinical trial of spinal cord injury. The metabolic product of TRH (cyclo-his-pro) retains physiological activity. We have developed a number of novel cyclic dipeptides that are structurally similar to cyclo-his-pro, and have examined their neuroprotective activity across multiple in vitro models of neuronal injury and after traumatic brain injury (TBI) in rodents. Four such compounds were found to reduce cell death after trophic withdrawal or traumatic injury in primary neuronal cultures; two also protected against glutamate or beta-amyloid neurotoxicity. All compounds significantly improved motor and cognitive recovery after controlled cortical impact injury in mice, and markedly reduced lesion volumes as shown by high field magnetic resonance imaging. Further, compound 35b, which is being developed for clinical trials, also showed considerable neuroprotection after fluid percussion induced TBI in rats, and improved cognitive function after daily administration in chronically brain injured rats. At a mechanistic level, the drugs attenuate both apoptotic and necrotic cell death in primary neuronal cultures, markedly reduce intracellular calcium accumulation after injury, and limit changes in mitochondrial membrane potential and associated cytochrome c release. In addition, microarray studies show that 35b reduces transcriptional changes after injury for a number of genes (and proteins) that may be associated with secondary injury, including cell cycle genes, aquaporins and cathepsins. It also upregulates brain-derived neurotrophic factor (BDNF), heat shock proteins (HSP) and hypoxia inducible factor (HIF). Thus, these novel dipeptides have multipotential actions that make them candidates for the treatment of both acute and chronic neurodegeneration.

The use of single-donor fibrin glue prepared by recycled cryoprecipitation in experimental liver surgery.

The purpose of the study was to evaluate the hemostatic effectiveness of fibrin glue (FG) prepared by a modification of cryoprecipitation technique in experimental rat liver surgery. FG component 1 was prepared by triple or 'recycled' cryoprecipitation method from single-donor plasma. Rats subjected to liver incision, partial and total lobectomy were treated with FG on the surgical cut surface or underwent standard surgical technique. The efficacy of FG-treatment was evaluated on the basis of the 24-hour survival ratio and peripheral blood hematological parameters. The mean values of fibrinogen, FXIII, fibronectin and horizontal tensile strength of FG were 54.2 +/- 19.9 g/l, 13.5 +/- 3.6 IU/ml, 3103.1 +/- 148.9 mg/l, and 1.076 +/- 0.18 N/cm2, respectively. The survival of FG-treated rats subjected to partial and total lobectomy was significantly higher in comparison to the FG-nontreated animals, accompanied with higher values of red blood cell counts, hemoglobin concentration and hematocrit. When liver incision was performed, although there were no differences in survival rate, FG-treated animals had significantly higher values of the tested hematological parameters. The presented results demonstrated that by using 'recycled' cryoprecipitation it is possible to obtain high quality single-donor FG with successful hemostatic therapeutical effects, as confirmed in the experimental rat model of liver surgery.

Temporal characterisation of pro- and anti-apoptotic mechanisms following diffuse traumatic brain injury in rats.

Few studies have characterised apoptosis in a brain injury model that causes a significant degree of diffuse axonal injury. Such characterisation is essential from a clinical viewpoint since diffuse axonal injury is a major component of human head injury. The present study therefore, examines the expression of active and proactive caspase-3, and the bax, bcl-2 and bcl-x members of the bcl-2 family, to characterise the temporal profile of apoptosis in a model of traumatic brain injury in rats that produces significant diffuse axonal injury. Pentobarbital anaesthetised male Sprague-Dawley rats were injured using the 2m impact-acceleration model of diffuse traumatic brain injury. After injury, diffuse trauma resulted in an increased bax expression followed by induction of caspase-3. The increase in caspase-3 was simultaneous with an increase in anti-apoptotic bcl-2 expression. Bcl-x levels were increased after induction of caspase-3 and the increased levels of bcl-x were sustained to the end of the 5-day observation period. Increased active caspase-3 expression was associated with the appearance of TUNEL positive cells. These cells were detected in different brain regions at different times, with some regions showing no apoptotic cells until 3 days after injury. No TUNEL positive cells were detected at 7 and 14 days after injury. DNA electrophoresis confirmed that DNA fragmentation was maximal at 3 days after injury. Increased active caspase-3 levels were also significantly correlated with increased bcl-2 levels (r=0.80; P<0.001) suggesting that the apoptotic cascade after diffuse traumatic brain injury is a carefully controlled cellular homeostatic response. Pharmacological manipulation of this balance may offer a therapeutic approach for preventing cell death and improving outcome after diffuse traumatic brain injury.