Allopregnanolone and progesterone decrease cell death and cognitive deficits after a contusion of the rat pre-frontal cortex.

We compared the effects of three different doses of allopregnanolone (4, 8 or 16 mg/kg), a metabolite of progesterone, to progesterone (16 mg/kg) in adult rats with controlled cortical impact to the pre-frontal cortex. Injections were given 1 h, 6 h and every day for 5 consecutive days after the injury. One day after injury, both progesterone-treated (16 mg/kg) and allopregnanolone (8 or 16 mg/kg)-treated rats showed less caspase-3 activity, and rats treated with allopregnanolone (16 mg/kg) showed less DNA fragmentation in the lesion area, indicating reduced apoptosis. Nineteen days after the injury, rats treated with progesterone and allopregnanolone (8 or 16 mg/kg) showed no difference in necrotic cavity size but had less cell loss in the medio-dorsal nucleus of the thalamus and less learning and memory impairments compared with the injured vehicle-treated rats. On that same day the injured rats treated with progesterone showed more weight gain compared with the injured rats treated with the vehicle. These results can be taken to show that progesterone and allopregnanolone have similar neuroprotective effects after traumatic brain injury, but allopregnanolone appears to be more potent than progesterone in facilitating CNS repair.

Serum progesterone levels correlate with decreased cerebral edema after traumatic brain injury in male rats.

Previous animal research suggests that progesterone may have powerful neuroprotective effects in traumatic brain injury (TBI). This experiment tested the hypothesis that progesterone levels correlate with decreased cerebral edema in male rats with bilateral medial frontal cortex injuries. Three groups of male Sprague-Dawley rats were used: injured given progesterone (4 mg/kg), injured given vehicle (oil), and uninjured controls given vehicle. Progesterone or vehicle was administered intraperitoneally at 1, 6, and 24 h postinjury. At 48 h postinjury, the rats were killed, brains extracted, and assayed for edema. Percent difference in water content of the area surrounding the lesion was compared to posterior cortex. A strong inverse relationship was found between serum progesterone levels and percent cerebral edema; the higher the progesterone levels, the lower the percent edema. Both progesterone and oil-treated animals had some edema compared to sham-operated controls. The brains of the injured animals given control solution were higher in water content than either the uninjured group or injured progesterone-treated rats 48 h postinjury. These findings confirm that progesterone significantly decreases cerebral edema after TBI in adult male subjects.

Biocompatibility of methylcellulose-based constructs designed for intracerebral gelation following experimental traumatic brain injury.

Tissue engineering in the post-injury brain represents a promising option for cellular replacement and rescue, providing a cell scaffold for either transplanted or resident cells. We have characterized the use of methylcellulose (MC) as a scaffolding material, whose concentration and solvent were varied to manipulate its physical properties. MC solutions were produced to exhibit low viscosity at 23 degrees C and form a soft gel at 37 degrees C, thereby making MC attractive for minimally invasive procedures in vivo. Degradation and swelling studies in vitro demonstrated a small amount of initial polymer erosion followed by relative polymer stability over the 2-week period tested as well as increased hydrogel mass due to solvent uptake. Concentrations up to 8% did not elicit cell death in primary rat astrocytes or neurons at 1 or 7 days. Acellular 2% MC (30 microl) was microinjected into the brains of rats 1 week after cortical impact injury (velocity = 3 m/s, depth = 2 mm) and examined at 2 days (n = 8; n = 3, vehicle injected) and 2 weeks (n = 5; n = 3, vehicle injected). The presence of MC did not alter the size of the injury cavity or change the patterns of gliosis as compared to injured, vehicle-injected rats (detected using antibodies against GFAP and ED1). Collectively, these data indicate that MC is well suited as a biocompatible injectable scaffold for the repair of defects in the brain.

Effects of the duration of progesterone treatment on the resolution of cerebral edema induced by cortical contusions in rats.

UNLABELLED: PURPOSE. The aim of the present study was to assess the effect of different durations of administration of progesterone (4 mg/kg) on the resolution of edema 6 days after medial frontal cortex contusions (MFC) in male adult rats. METHODS: Animals sustaining injury were injected with progesterone or its vehicle for 3 days or for 5 days beginning the first hour after surgery. On the 6th day the rats were killed and their brain water content was measured. RESULTS: We confirmed the presence of edema six days after MFC. However, both 3 and 5 days of treatment with progesterone significantly reduced edema in the injured brains but the five days of treatment were more effective. The effects of progesterone depend upon the duration of the treatment because there are two waves of edema. The first phase begins within a few hours of the injury and the second starts several days later. CONCLUSIONS: Our data are consistent with earlier findings showing that longer durations of progesterone administration lead to more complete behavioral recovery as well as to an increased number of surviving neurons.

Astrocytes generate isoprostanes in response to trauma or oxygen radicals.

Previous studies have shown that oxygen radical scavengers prevent the reduced cerebral blood flow that occurs following experimental traumatic brain injury. The exact chemical species responsible for the posttraumatic reduction in flow is unknown. We tested whether isoprostanes, which are formed by non-cyclooxygenase-dependent free radical attack of arachidonic acid and are vasoconstrictors of the cerebral circulation, are increased in astrocytes following stretch-induced trauma or injury with a free radical generating system. Isoprostane (8-epi-prostaglandin F2alpha) was analyzed in cells and in media by immunoassay. Confluent rat cortical astrocytes in culture were injured by a hydroxyl radical generating system consisting of hydrogen peroxide and ferrous sulfate or by rapid stretch of astrocytes grown on a deformable silastic membrane. Some cells were treated with the iron chelator deferoxamine for 1 h before injury. The hydroxyl generating system caused free and cell-bound isoprostanes to increase to more than 400% of control. After trauma, free and membrane bound isoprostanes increased to 321 +/- 34% and 229 +/- 23% of control, respectively, and posttraumatic increases were prevented by deferoxamine. Since astrocytes are in close proximity to cerebral vessels, posttraumatic free radical formation may increase the formation of isoprostanes, which in turn produce vasoconstriction and decrease cerebral blood flow.

Calcium influx factor, further evidence it is 5, 6-epoxyeicosatrienoic acid.

We present evidence in astrocytes that 5,6-epoxyeicosatrienoic acid, a cytochrome P450 epoxygenase metabolite of arachidonic acid, may be a component of calcium influx factor, the elusive link between release of Ca2+ from intracellular stores and capacitative Ca2+ influx. Capacitative influx of extracellular Ca2+ was inhibited by blockade of the two critical steps in epoxyeicosatrienoic acid synthesis: release of arachidonic acid from phospholipid stores by cytosolic phospholipase A2 and cytochrome P450 metabolism of arachidonic acid. AAOCF3, which inhibits cytosolic phospholipase A2, blocked thapsigargin-stimulated release of arachidonic acid as well as thapsigargin-stimulated elevation of intracellular free calcium. Inhibition of P450 arachidonic acid metabolism with SKF525A, econazole, or N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide, a substrate inhibitor of P450 arachidonic acid metabolism, also blocked thapsigargin-stimulated Ca2+ influx. Nano- to picomolar 5, 6-epoxyeicosatrienoic acid induced [Ca2+]i elevation consistent with capacitative Ca2+ influx. We have previously shown that 5, 6-epoxyeicosatrienoic acid is synthesized and released by astrocytes. When 5,6-epoxyeicosatrienoic acid was applied to the rat brain surface, it induced vasodilation, suggesting that calcium influx factor may also serve a paracrine function. In summary, our results suggest that 5,6-epoxyeicosatrienoic acid may be a component of calcium influx factor and may participate in regulation of cerebral vascular tone.

Progesterone protects against lipid peroxidation following traumatic brain injury in rats.

The gonadal hormone, progesterone, has been shown to have neuroprotective effects in injured nervous system, including the severity of postinjury cerebral edema. Progesterone's attenuation of edema is accompanied by a sparing of neurons from secondary neuronal death and with improvements in cognitive outcome. In addition, we recently reported that postinjury blood-brain barrier (BBB) leakage, as measured by albumin immunostaining, was significantly lower in progesterone treated than in nontreated rats, supporting a possible protective action of progesterone on the BBB. Because lipid membrane peroxidation is a major contributor to BBB breakdown, we hypothesized that progesterone limits this free radical-induced damage. An antioxidant action, neuroprotective in itself, would also account for progesterone's effects on the BBB, edema, and cell survival after traumatic brain injury. To test progesterone's possible antiperoxidation effect, we compared brain levels of 8-isoprostaglandin F2 alpha (8-isoPGF2 alpha), a marker of lipid peroxidation, 24, 48, and 72 h after cortical contusion in male rats treated with either progesterone or the oil vehicle. The brains of progesterone treated rats contained approximately one-third of the 8-isoPGF2 alpha found in oil-treated rats. These data suggest progesterone has antioxidant effects and support its potential as a treatment for brain injury.

Synaptic remodeling and free radical formation after brain contusion injury in the rat.

The purpose of this study was to explore whether bilateral frontal cortex contusion in rats would demonstrate changes relevant for understanding the pathology of frontal lobe injury in humans. Rats were allowed to survive for 3, 7, or 18 days postinjury (dpi). In the contused rats, albumin was trapped in frontal cortices, as well as in other brain areas, showing that neurons were exposed to plasma components. In the sham-operated rats, which had only craniotomy but no penetration of dura, the level of trapped albumin was also increased compared to intact controls, suggesting a partial lesion-like condition. Choline acetyltransferase activity was severely decreased in the frontal cortices of contused rats, compared to the sham-operated controls. The decrease was most pronounced at 3 dpi and less pronounced 18 dpi, suggesting that after the initial damage, regeneration of the cholinergic terminals occurred. The concentration of the mature presynaptic membrane protein D3(SNAP-25) was also decreased in the frontal cortices of contused rats at 3 and 7 dpi, whereas it was normalized at 18 dpi. Previously, we have evaluated changes in the rate of synaptic remodeling in brain injury by calculating the ratio of the neural cell adhesion molecule (NCAM) to D3(SNAP-25). The NCAM/D3(SNAP-25) ratio at 3 dpi was elevated by more than 60% in the frontal cortices of contused rats, suggesting a high initial rate of synaptic remodeling. The ratios were smaller at 7 and 18 dpi, suggesting that after the initial burst, the rate of remodeling leveled off. In contrast, astrocyte activation was less pronounced at 3 dpi than at 7 and 18 dpi, as measured by the levels of glial fibrillary acidic protein and glutamine synthetase immunoactivities. The immunoreactivity of glutamine synthetase more than doubled in the contused brains but its enzymatic activity increased less than 50%, suggesting that many enzymatic centers had been inactivated by free radicals. Calculated as the difference between the relative immunoreactivity and the relative enzymatic activity the "lost glutamine synthetase activity" increased continuously in frontal cortex and striatum from 3 to 18 dpi, indicating the production of free radicals long after the initial contusion event. In conclusion, following frontal cortical contusions the early synaptic damage was partly compensated by synaptic remodeling. We suggest that the continuous production of free radicals may have contributed to the declining remodeling rate and impair functional recovery.

Isoprostanes: free radical-generated prostaglandins with constrictor effects on cerebral arterioles.

BACKGROUND AND PURPOSE: Isoprostanes are generated by cyclooxygenase-independent free radical attack of arachidonic acid and are potent constrictors of the peripheral vasculature. Traumatic brain injury stimulates oxygen radical production and is associated with cerebral blood flow reduction. However, no specific vasoconstrictor has been identified as the cause of posttraumatic blood flow reduction. The purpose of this study was to determine whether isoprostanes constrict cerebral arterioles. METHODS: The effects of 10(-9) to 10(-5) mol/L 8-iso-prostaglandin F2 alpha (8-iso-PGF2 alpha), 8-iso-prostaglandin E2 (8-iso-PGE2), and prostaglandin F2 alpha (PGF2 alpha) on pial arteriolar diameter were measured in anesthetized rats using a closed cranial window and in vivo microscopy. RESULTS: All prostanoids produced vasoconstriction. Of these, 8-iso-PGF2 alpha produced the greatest vasoconstriction (34% +/- 2), followed by 8-iso-PGE2 (25% +/- 4) and PGF2 alpha (20% +/- 2). After six cerebrospinal fluid washouts of the cranial window, both 8-iso-PGF2 alpha- and 8-iso-PGE2-treated vessels remained slightly constricted, whereas the PGF2 alpha-treated vessels returned to control diameter. Coapplication of the semiselective thromboxane A2/prostaglandin H2 receptor antagonist SQ29548 completely blocked the vasoconstriction induced by 8-iso-PGF2 alpha and 8-iso-PGE2. CONCLUSIONS: Isoprostanes are potent constrictors of cerebral arterioles and appear to act at a receptor that is similar to the thromboxane A2/prostaglandin H2 receptor. Isoprostanes may play a role in the reduction of cerebral blood flow that occurs after brain injury and subsequent oxygen radical production.

Extract of Ginkgo biloba (EGb 761) improves behavioral performance and reduces histopathology after cortical contusion in the rat.

Male rats received bilateral frontal cortex contusions and were injected with 100 mg/kg of EGb 761 or an equal volume of vehicle beginning 5 min after injury and then with 1 injection/day for 7 days. The rats were tested for spontaneous motor behavior on days 1, 5, 10, and 15 postinjury and then for 10 days of spatial navigation performance in the Morris Water Maze (MWM), beginning on the day 8 after the contusion. Brain tissue was removed for examination on the 18th day after injury. Contused rats given EGb 761 performed more like intact rats on measures of spontaneous motor activity while vehicle-treated counterparts remained more active than either shams or EGb 761-treated animals by the conclusion of testing. Contusion-only rats were worse than shams on spatial performance, while those given EGb 761 were less impaired. Histological analyses indicated that EGb 761 failed to prevent loss of tissue at the primary site of impact. However, the extract reduced retrograde degeneration of neurons, gliosis in the thalamus, and ex vacuo hydrocephalus. EGb 761 treatment also decreased the loss of ChAT-positive neurons in the dorsomedial caudate-putamen and in the nucleus basalis magnocellularis (NBM). The results of this study indicate that EGb 761 could be a possible treatment for traumatic brain injury.

A reliable and sensitive enzyme immunoassay method for measuring 8-isoprostaglandin F2 alpha: a marker for lipid peroxidation after experimental brain injury.

The objectives of this study were to determine (1) if levels of 8-isoprostaglandin F2 alpha (8-isoPGF2 alpha), a non-enzymatically produced prostaglandin, were increased after cortical contusion and (2) if enzyme immunoassay (EIA) could be used to quantify 8-isoPGF2 alpha levels. 24 h after the contusion there was a significant rise in 8-isoPGF2 alpha compared to control levels. The levels returned to baseline by 72 h postinjury. The results show that this EIA method is a reliable and sensitive technique for measuring brain lipid peroxidation.

Blood-brain barrier breakdown and edema formation following frontal cortical contusion: does hormonal status play a role?

The present experiment was designed to evaluate and correlate the time course of blood-brain barrier (BBB) integrity and cerebral edema in adult male rats given medial frontal cortex contusions. The effect of sex hormones on BBB integrity in the same injury model was also examined, because previous work has shown that progesterone can reduce cerebral edema (Roof et al., 1993). BBB breakdown was assessed by Evans blue extravasation and albumin immunostaining while edema formation was measured by the wet weight dry weight technique. These processes were examined beginning 2 h and continuing up to 10 days after injury. Our findings show that medial frontal contusion in rats produces changes in cerebral water content and opening of the BBB that endures at least 7 days postinjury. Although pseudopregnancy has been shown to reduce cerebral edema at day 1 postinjury, we did not find any evidence that this hormonal state is associated with BBB repair.

Bilateral frontal cortical contusion in rats: behavioral and anatomic consequences.

The purpose of this study was to develop a bilateral model of frontal cortical contusion in the rat that would demonstrate reproducible deficits typically found after frontal lobe injury in humans. We used a pneumatically controlled cortical impactor to create bilateral contusions of the medial prefrontal cortex (PFC) in adult male Sprague-Dawley rats. Cognitive, neurologic, physiologic, and histopathologic measures were used to evaluate changes caused by the injury. The cognitive task employed the Morris water maze (MWM). Contused rats performed worse than sham-operated controls on measures of time taken to find a submerged platform, distance to the platform, and swim strategy. Neurologic measures revealed impairments of tongue mobility and transient deficits of forelimb placing. Body weights of the contused rats were chronically reduced with respect to controls, indicating that cortical contusion produces disruption in homeostasis. All rats given bilateral PFC contusions developed marked necrotic cavities at the site of impact. The borders surrounding the cavities were heavily lined with astrocytes and ameboid microglia. There was subcortical gliosis in the medial caudate that extended throughout the rostral-caudal length of the caudate-putamen and into the mediodorsal (MD) and ventrolateral (VL) nuclei of the thalamus. The thalamus was also the site of distal transneuronal degeneration. In both the MD and the VL, there was significant neuronal loss in the contused rats as compared with sham-operated controls. This method of bilateral cortical contusion demonstrates clear, reproducible results that would be required for the development of future pharmacologic therapies designed to promote functional recovery.

Vaginocervical stimulation attenuates hindpaw shock-induced substance P release into spinal cord superfusates in rats.

The present study was designed to elucidate the mechanism in the spinal cord by which vaginocervical stimulation (VS) attenuates responses to noxious stimulation. This was accomplished by testing the hypothesis that VS reduces noxious stimulation-induced release of substance P at the level of the spinal cord. Noxious foot shock significantly increased the release of substance P (measured using radioimmunoassay) into superfusates of the lumbosacral spinal cord region in urethane-anesthetized rats. VS applied concurrently with foot shock significantly attenuated the release of substance P compared to the foot shock-only condition. In addition, substance P levels were significantly lower after the VS-only condition than after the no stimulation or foot shock-only conditions. These findings indicate that VS may produce analgesia, at least in part, by suppressing the release of substance P within the spinal cord.

Conceptual and practical issues in the pharmacological treatment of brain injury.

It is only within the last ten years that research on treatment for central nervous system (CNS) recovery after injury has become more focused on the complexities involved in promoting recovery from brain injury when the CNS is viewed as an integrated and dynamic system. There have been major advances in research in recovery over the last decade, including new information on the mechanics and genetics of metabolism and chemical activity, the definition of excitotoxic effects and the discovery that the brain itself secretes complex proteins, peptides and hormones which are capable of directly stimulating the repair of damaged neurons or blocking some of the degenerative processes caused by the injury cascade. Many of these agents, plus other nontoxic naturally occurring substances, are being tested as treatment for brain injury. Further work is needed to determine appropriate combinations of treatments and optimum times of administration with respect to the time course of the CNS disorder. In order to understand the mechanisms that mediate traumatic brain injury and repair, there must be a merging of findings from neurochemical studies with data from intensive behavioral testing.

How providers can preserve mutually beneficial joint ventures.

Joint ventures between hospitals and physicians were formed at a dizzying pace during the 1980s. Many of these joint ventures are now a source of concern for both hospital and physician partners. In the following article, the authors discuss their experience with joint ventures in the wake of recent and proposed regulatory and legislative activity and present several practical strategies for hospital executives to consider regarding their joint ventures.

Effects of BIM-22015, an analog of ACTH4-10, on functional recovery after frontal cortex injury.

Male rats, 90-100 days old, with frontal cortex lesions were given either subcutaneous sterile water (SW) as a vehicle control or 1, 10, or 100 micrograms of BIM-22015 every other day for 20 days. Brain-injured subjects tested in the Morris water maze with either 10 micrograms BIM-22015 or SW took significantly more trials than sham-operated rats to locate a submerged platform eight consecutive times within 60 s. The animals given 1 or 100 micrograms BIM-22015 took significantly fewer trials to reach criterion than brain-injured animals in the other drug treatment groups. On a percentage of savings, measured 8 days after reaching criterion, the brain-injured subjects given 1, 10, or 100 micrograms BIM-22015 did not differ from sham-operated rats. In contrast, the brain-injured animals given SW took longer to find the submerged platform than they did during the initial training. To assess long-term effects of the ACTH analog treatment, rats were trained on a delayed spatial alternation task 30 days after receiving the last injection. On this task, brain-injured rats treated with the 10-micrograms dose performed significantly better than those given sterile water. Acetylcholinesterase (AChE)-labeled neurons counted in the nucleus basalis magnocellularis indicated that rats with frontal cortex damage given the 10-micrograms treatment did not differ from the sham controls and had significantly more AChE-positive neurons than injured counterparts treated with SW or 100 micrograms.

Intracerebral administration of alpha-tocopherol-containing liposomes facilitates behavioral recovery in rats with bilateral lesions of the frontal cortex.

Adult rats with bilateral frontal cortex lesions received intracerebral infusion of phosphatidylcholine liposomes, or D-alpha-tocopherol-enriched liposomes, delivered continuously for 7 days to the damaged cortex by subcutaneous osmotic pumps. All subjects were first tested on a delayed spatial alternation task and then, 90 days later, on a spatial navigation task in the Morris water maze. Both tests showed that brain-injured rats with alpha-tocopherol treatment were less impaired than counterparts treated with plain liposomes. Alpha-tocopherol also reduced some of the injury-induced, secondary reactive changes that typically follow damage to the frontal cortex.

Ginkgo biloba extract facilitates recovery from penetrating brain injury in adult male rats.

Adult, male Sprague-Dawley rats received 100 mg/kg Ginkgo biloba extract (GBE) intraperitoneally for 30 days. GBE reduced overall activity and decreased sensitivity to light in the open field maze. The rats were also less responsive to noxious stimuli after 13 days of treatment with GBE. After the last injection, all subjects were trained on a delayed-spatial alternation task. Subsequent to acquisition of the spatial task, the rats received either sham operations and saline or bilateral frontal cortex lesions treated with either saline or GBE. Thirty additional days of treatment began on the day of injury, and open field behavior, analgesia, and metabolic activity measurements were again measured. The rats with lesions treated with saline were more active than their GBE-treated counterparts and sham controls but there were no differences in response to illumination or noxious stimuli. Retention of the delayed-spatial alternation indicated that rats with lesions treated with GBE were less impaired than brain-injured subjects receiving saline treatment. Histological examination showed that GBE reduced the extent of brain swelling in response to the injury.

Clinal genetic variation at enzyme loci in bald eagles (Haliaeetus leucocephalus) from the western United States.

Five polymorphic enzyme loci of about 50 sampled were discovered in blood extracts of bald eagles from Alaska, Washington, Oregon, and Arizona, representing the first biochemical genetic variation described for the species. All five loci exhibited trends of north-to-south clinal geographic variation in gene frequencies. Gene frequencies at three loci culminated in fixation in the Arizona population, which consists of 12 known breeding pairs. The Arizona birds were maximally heterozygous at the other two loci, suggesting the possibility of maintenance of some clines by natural selection. No significant discontinuities in gene frequencies were observed which correlated with earlier descriptions of two subspecies (northern and southern races) of bald eagles.