Effect of dimethyl sulfoxide on blood-brain barrier integrity following middle cerebral artery occlusion in the rat.

Dimethyl sulfoxide (DMSO) is widely used as a solvent for other drugs, i.e., for the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) and the V1a receptor-antagonist SR49059, to reduce brain edema. We studied the effect of DMSO on blood-brain barrier (BBB) integrity following middle cerebral artery occlusion (MCAO) and the consequences on brain edema development. Male Sprague-Dawley rats were randomly assigned to sham procedure or infusion of 1% DMSO, PMA (230 microg/kg in 1% DMSO), or SR49059 (1 mg/kg in 1% DMSO) followed by MCAO (each group n = 10). After a 2-hour period of ischemia and 2 hours reperfusion, the animals were sacrificed for assessment of brain water content, sodium, and potassium concentration. BBB integrity was assessed by Evans blue extravasation. Statistical analysis was performed by ANOVA followed by a Tukey post hoc test. Low-dose DMSO treatment following MCAO significantly opened the BBB on the ischemic side (p < 0.037). PMA and SR49059 did not have any additional effect on BBB compromise compared to DMSO (p = 1.000, p < 0.957, respectively). We conclude that DMSO as a vehicle for drug administration may increase the drug concentration into the extracellular space, but since BBB permeability is increased, it may also provide an avenue for development of vasogenic edema.

Protective effect of the V1a receptor antagonist SR49059 on brain edema formation following middle cerebral artery occlusion in the rat.

There exists no pharmacological treatment for fulminating brain edema. Since evidence indicates that brain aquaporin-4 (AQP4) water channels are modulated by vasopressin V1a receptors, we examined the edema-reducing properties of the selective V1a receptor antagonist, SR49059, following middle cerebral artery occlusion (MCAO). Male Sprague-Dawley rats were randomly assigned to sham procedure, vehicle, or SR49059 infusion at different dosages (each n = 6,480 microL/hr, 640 microL/hr, 720 microL/hr) and starting 60 minutes before or after MCAO. After a 2-hour period of ischemia and 2 hours of reperfusion, the animals were sacrificed for assessment of brain water content, sodium, and potassium concentration. Statistics were performed using an ANOVA followed by a Tukey post hoc analysis. SR049059 treatment reduced brain water content in the infarcted area given at 640 microL/hr (p = 0.036), 720 microL/hr 60 minutes before (p = 0.002) or 60 minutes after (p = 0.005) MCAO. The consecutive sodium shift into the brain was prevented (p = 0.001), while the potassium loss was inhibited only by pre-treatment (p = 0.003). These findings imply that in ischemia-induced brain edema, the selective V1a receptor-antagonist SR49059 inhibits brain edema and the subsequent sodium shift into brain. This substance offers a new avenue in brain edema treatment and prompts further study into AQP4 modulation.

Modulation of AQP4 expression by the protein kinase C activator, phorbol myristate acetate, decreases ischemia-induced brain edema.

The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), is known to interact with aquaporin-4 (AQP4), a water-selective transporting protein abundant in astrocytes and ependymal cells, that has been found to decrease osmotically-induced swelling. The purpose of this study was to examine whether PMA given at different time points following focal ischemia induced by middle cerebral artery occlusion (MCAO) reduces brain edema by AQP4 modulation. Male Sprague-Dawley rats were randomly assigned to sham procedure, vehicle, or PMA infusion (230 microg/kg), starting either 60 minutes before, or 30 or 60 minutes after MCAO (each group n = 12). After a 2-hour period of ischemia and 2 hours of reperfusion, the animals were sacrificed for assessment of brain water content, sodium, and potassium concentrations. AQP4 expression was assessed by immunoblotting. Statistical analysis was performed by ANOVA followed by Tukey's post hoc test. PMA treatment significantly reduced brain water content concentration in the infarcted area when started before or 30 minutes post-occlusion (p < 0.001, p = 0.022) and prevented the subsequent sodium shift (p < 0.05). Furthermore, PMA reduced ischemia-induced AQP4 up-regulation (p < 0.05). Attenuation of the ischemia-induced AQP4 up-regulation by PMA suggests that the reduction in brain edema formation following PMA treatment was at least in part mediated by AQP4 modulation.

Modulation of aquaporin-4 water transport in a model of TBI.

Our Laboratory has pursued the hypothesis that traumatic brain edema is predominantly cellular and recent supportive evidence has been obtained indicating a non-extracellular route for sodium and water entering brain. The aim of this study was to investigate if astrocytic endfeet are involved in this passage, using a potent activator of Protein Kinase C (phorbol ester) to modify and closing the Aquaporin 4 (AQP4), a water channel specific for astrocytic endfoot. Anaesthetized Sprague-Dawley rats were subjected to an intracerebroventricular bolus of phorbol ester (50 pmol/4 microl) or vehicle, in the right hemisphere and after 30 minutes they were exposed to the well-established conical contusion model (3 mm depth at 6 m/sec) on the same side. After trauma, they were subjected to 5 hours of drug continuous infusion, then sacrificed. Water content measurements for both right (injured) and left (uninjured) hemispheres were calculated using the wet weight/dry weight technique. Results of these experiments showed a significant decrease in water content in injured phorbol treated animals, underlying that AQP4 regulation plays an important role in brain edema following stroke, and supporting the concept of cellular formation for edema via astrocytic foot processes.

The effect of cyclosporin A on brain edema formation following experimental cortical contusion.

Cyclosporin A (CsA) has been shown by our laboratory and others to be neuroprotective in the experimental animal model of traumatic brain injury. However, we found that the intrathecal administration resulted in a concomitant increase of brain edema. The aim of this study was to assess whether intravascular administration may also influence brain edema formation. This project includes two independent series in which different doses of CsA were intravenously given to Sprague-Dawley rats of each group. In the first series, the animals were exposed to focal brain injury by a controlled cortical impact (CCI, 6 m/sec, 3 mm depth) and randomized into the following two groups: 20 mg/kg CsA and control vehicle. In the second series, animals were also injured by CCI and randomized into 35 mg/kg CsA and control vehicle. The intravenous continuous (1 h) infusion was begun 30 minutes after the insult. All animals were sacrificed at 24 hours post injury to assess the brain water content using the gravimetric method. Intravenously-administrated CsA of either 20 or 35 mg/kg did not significantly change the brain water content. We therefore suggest that an intravascular route may be better for CsA administration because the intrathecal injection may exacerbate brain edema as found in our previous study.

Acute blood-brain barrier changes in experimental closed head injury as measured by MRI and Gd-DTPA.

The objective of this study was to determine the early time course of blood-brain barrier (BBB) changes in diffuse closed head injury (CHI) and to what extent BBB is affected by secondary insult. The BBB disruption was quantified using T1-weighted MRI following administration of Gd-DTPA. The maximal signal intensity (SI) enhancement was used to calculate BBB disruption. A new CHI model was used to induce injury. Adult SD rats were separated into four groups: Group I: Sham (n = 4), II: Hypoxia and Hypotension (HH, n = 4), III: Trauma alone (n = 23), and IV: Trauma coupled with HH (THH, n = 14). Following trauma, a 30 minute insult of hypoxia (PaO2 = 40 mmHg) and hypotension (MABP = 30 mmHg) were imposed. In trauma animals, SI increased dramatically immediately following impact. By 15 minutes, permeability decreased exponentially and by 30 minutes was equal to that of control. In THH animals, SI enhancement was lower after the trauma, consistent with reduced blood pressure and blood flow. However, the SI increased dramatically upon reperfusion and was equal to that of control after 60 minutes. In conclusion we may consider, that CHI is associated with a rapid and transient BBB opening which begins at the time of the trauma and lasts not more than 30 minutes. It has been also shown that addition of hypoxia and hypotension prolongs the time of BBB breakdown.

Coma associated with flaccidity produced by fluid-percussion concussion in the cat. I: Is it due to depression of activity within the brainstem reticular formation?

This study is the first attempt to characterize neurological and behavioural consequences of fluid-percussion concussive head injury in the cat. Both animals initially anaesthetized by N2O as well as unanaesthetized, chronically prepared animals were subjected to injury. Injury with a fluid-pressure wave of 1.9-2.5 atm (duration 21-24 ms) produced a brief generalized areflexia. Following this initial response, injury greater than 2.1 atm frequently produced a period associated with hypotonia of postural muscles and suppression of postural motor responses (flaccidity). A close association between flaccidity and other indices of coma such as absence of eye-opening responses was noted. These consequences of injury can occur without fatal apnoae, circulatory collapse or overt intraparenchymal haemorrhages. This result suggests that mechanical stress predominantly restricted to the brain stem in fluid percussion may be sufficient, at least in the cat, to produce coma associated with flaccidity which has been previously documented for acceleration concussion. There was no evidence that fluid percussion produced EEG depression similar to the effects of lesions in the mesencephalic reticular activating system (RAS). Thus, depression of general levels of brain activity including those within the RAS seems not be necessary for production of this form of reversible coma.

Coma associated with flaccidity produced by fluid-percussion concussion in the cat. II: Contribution of activity in the pontine inhibitory system.

In the preceding paper we reported that concussive levels of fluid-percussion head injury can produce transient flaccidity of postural muscles associated with other indices of coma. This reversible coma associated with flaccidity follows an initial period of generalized areflexia and occurs in the absence of EEG slow waves. The present study investigated the physiological mechanisms underlying the flaccidity following concussive head injury be recording dorsal and ventral root potentials of the spinal cord. Studies indicated that, during the initial period of generalized areflexia, afferent input transmission was depressed although the excitability of motoneuronal pools was increased. In contrast, during periods of flaccidity, spinal cord somatomotor functions were depressed while transmission of afferent inputs was recovering. Systematic transection of the brain stem showed that activity within structures lying between collicular and midpontine levels is necessary to produce this latter condition. Cholinergic activation of pontine inhibitory areas within this same region of the rostral pons can produce profound descending inhibitory influences on postural somatomotor function in conjunction with other features of coma including suppression of eye-opening responses. Such effects occur without EEG slow waves. Moreover, other data indicate that local rates of glucose utilization within this pontine inhibitory area increase following concussive head injury. Thus, it is possible that a predominance of activity within the pontine inhibitory area could provide at least one neural basis for the reversible comatose state following concussive head injury characterized by close association between flaccidity and other indices of coma. Possible relationships of these data to clinically observed features of concussion are discussed.