INTRODUCTION

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SINCE 1965, VARIOUS RESEARCHERS have investigated hyperbaric oxygen (HBO) to attenuate cerebral ischemia in animal models and in humans. Takahashi et al. (18) found that HBO therapy [in a canine model of complete global cerebral ischemia; 15 min HBO treatment at 3 atm absolute (ATA) for 1 h during the early postischemic period and 3 h after ischemia] accelerated neurologic recovery and improved the survival rate of dogs. Shiokawa et al. (17) reported that HBO therapy administered 3 h after inducing permanent incomplete brain ischemia (by ligating the common carotid artery) significantly increased the survival time of spontaneously hypertensive rats compared with the survival time of untreated ischemic animals. HBO (applied 2.5 and 3.5 h after occlusion) reduced ischemic neuronal injury, brain edema, and infarct area in a rat middle cerebral artery occlusion (MCAO) model (4 h without reperfusion) (9). In contrast, Shiokawa et al. (17) reported that HBO treatment did not show any therapeutic benefit when it was applied 1 h after cerebral ischemia in a permanent incomplete ischemia model in spontaneously hypertensive rats. Roos et al. (16) claimed that HBO therapy (2 ATA, 30 min) yielded no benefit in a rat model of MCAO and reperfusion.

We undertook this study to determine 1) whether HBO therapy yields a beneficial effect in an animal model of cerebral infarction induced through MCAO and reperfusion and 2) the optimal therapeutic window for starting HBO treatment for MCAO and reperfusion. The investigators in this study used a rat model of MCAO and reperfusion and evaluated the data resulting from HBO therapy (3 ATA HBO for 1 h) in the experimentally infarcted cerebral area.

	METHODS

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The Animal and Ethics Review Committee at the University of Mississippi Medical Center evaluated and approved the protocol used in this study.

Experimental groups. Forty-eight rats, each weighing 325-375 g, were divided into six treatment groups each consisting of eight subjects. The operator in this study performed the following procedures for each respective treatment group: 1) sham operation, exposure of the left common carotid artery and left external carotid artery followed by neither MCAO, reperfusion, nor HBO treatment; 2) MCAO for 2 h followed by reperfusion for 24 h; 3) MCAO for 2 h followed by reperfusion for 24 h and then HBO treatment applied at 3 h after reperfusion; 4) MCAO for 2 h followed by reperfusion for 24 h and then HBO treatment applied at 6 h after reperfusion; 5) MCAO for 2 h followed by reperfusion for 24 h and then HBO treatment applied at 12 h after reperfusion; 6) MCAO for 2 h followed by reperfusion for 24 h and then HBO treatment applied at 23 h after reperfusion.

MCAO model and HBO treatment. Forty-eight male Sprague-Dawley rats (assigned by study protocol to MCAO) underwent the procedure described by Longa et al. (10a). Accordingly, the operator anesthetized the rats with ketamine and xylazine and exposed the left common carotid artery. Then, the external carotid artery and its branches were isolated and coagulated. A 3-0 nylon suture with a blunted tip was inserted into the internal carotid artery through the external carotid artery stump and advanced to the anterior cerebral artery to occlude the middle cerebral artery (MCA). After occluding the MCA for 2 h, the operator carefully removed the suture to restore blood flow and then sutured the skin and allowed the rat to wake up. To complete the surgery, the operator applied 0.25% micaine locally to the wound and allowed the rat to recover.

Observing and evaluating the treated rats for neurological deficit. An experimenter, unaware of the treatments, tested the animals for neurological deficits after 24 h of reperfusion. Menzies et al. (11) developed the method that tests both motor and behavioral deficits on a cumulative scale from 0 to 4. This examination was used to evaluate ischemic injury: 0, no visible neurological deficits; 1, forelimb flexion; 2, contralateral forelimb grips weakly (the operator places the animal on an absorbent pad and gently pulls the tail); 3, circling to the paretic side only when pulled by the tail (the animal was allowed to move about freely on the absorbent pad); and 4, spontaneous circling.

Evaluation of infarcted area. After death had occurred, the coronal sections of the brain (2 mm thick) were cut and immersed in a 2% solution of 2,3,7-triphenyltetrazolium chloride. The stained slices were then fixed by immersion in phosphate-buffered 4% paraformaldehyde. The infarcted area and hemispheric area of each section were traced and measured using an image-analysis system (a Macintosh computer accessing the public domain National Institutes of Health Image program, written by Wayne Rasband and available from the internet). The percentage of infarction (infarct ratio) was calculated by dividing the infarcted area by the total area of the ipsilateral hemisphere (19).

Statistical analysis. Data were represented as the means ± SD. Statistical differences between the control (no HBO treatment) and the other groups were compared by using the one-way ANOVA and then Scheffé's F test if a significant difference was found; a P value <0.05 was considered statistically significant.

	RESULTS

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The study operator performed a pilot study to determine the MCAO model used to conduct this study. The surgical method and the size of the suture and its blunted tips were tested in different animals until a consistent neurological deficit (evaluated 24 h after reperfusion) was obtained. The rats used for this pilot study were not included among the 48 rats described by the above protocol. No neurological deficit was observed in the sham-operated rats.

Figure 1 presents the typical results of the effect of MCA occlusion on infarction size from each of the groups: the sham-operated (no MCAO), MCAO-no HBO treatment, and HBO at 3, 6, 12, and 23 h after reperfusion. The coronal sections were obtained by cutting the brain at a distance of 2, 4, 6, 8, and 10 mm from the rostral extremity of the frontal cortex. The white-colored areas represent the infarction regions in these sections.

View larger version (112K): [in this window] [in a new window]   Fig. 1.   Representative photographs of coronal sections of rat brains. From left to right are the sham-operated [no middle cerebral artery occlusion (MCAO)], MCAO-no hyperbaric oxygen (HBO) treatment, HBO at 3, 6, 12, and 23 h after reperfusion. The coronal sections were obtained by cutting the brain at a distance of 2, 4, 6, 8, and 10 mm from the top of frontal cortex (as shown from top to bottom). The white areas represent the infarct regions in these sections.

Figure 2 shows the results of the neurological deficit score in each group. Both the 3- and 6-h treatment groups exhibited a significantly improved neurological function (lower score) compared with the no-treatment group. In contrast, the neurological deficit was increased after HBO treatment at 12 and 23 h after reperfusion.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Neurological evaluation was performed according to the method of Menzies et al. (11). Animals were tested for neurological deficits by a blinded observer after 24 h of reperfusion. Grades of 0-4 were used. MCAO induced neurological deficits to an average level of grade 2.13 in rats without HBO treatment. HBO treatment for 1 h at 3 and 6 h after reperfusion reduced markedly the neurological scores. HBO treatment for 1 h at 12 and 23 h after reperfusion increased neurological scores. Eight rats were used in each group. *P < 0.05 (compared with no-HBO group, ANOVA).

Figure 3 shows the percentage of infarction area in rats that underwent MCAO and reperfusion and MCAO/reperfusion + HBO therapy at 3, 6, 12, and 23 h. No cerebral infarction was observed in the sham group. In the MCAO and reperfusion group (untreated group), severe cerebral infarction was observed in all rats and the infarct ratio was 11.34%. The infarct ratio was significantly decreased in rats treated with HBO at 3 (7.59%, P < 0.05 vs. untreated group) and 6 h (5.35%, P < 0.05 vs. untreated group) after reperfusion. However, the infarct ratio was significantly increased in rats treated with HBO at 12 (23%, P < 0.01 vs. untreated group) and 23 h after reperfusion (20%, P < 0.05 vs. untreated group).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   The infarct ratio was calculated as the %infarcted tissue per ispilateral hemisphere. In the no HBO-treated group, the infarct ratio is ~11.34% of the ispilateral hemisphere. HBO treatment at 3 and 6 h reduced markedly the infarct ratio. However, HBO treatment at 12 and 23 h enhanced infarct ratio. Eight rats were used in each group. * and **, P < 0.05 and 0.01, respectively (compared with no-HBO group, ANOVA).

	DISCUSSION

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The primary finding of this study is that the effect of HBO on the cerebral infarction is dual in an MCAO and reperfusion rat model. This finding suggests the existence of an optimal therapeutic window in which to initiate HBO therapy in this rat model of cerebral ischemia: early therapeutic intervention using HBO within 6 h reduces infarction. Applying HBO therapy late (after 12 h, for example) aggravates cerebral infarction; thus caution needs to be a consideration of HBO therapy in such cases.

Because current stroke treatment is unsatisfactory, many physicians seek novel treatment modalities. Because local anoxia and energy failures occur at the cellular level in ischemia, increasing the oxygen delivery to the tissues might prolong functional activity during severe ischemia (14). Animal studies and clinical experiences over the last two decades have produced a set of applications for which HBO therapy might appear beneficial (1). However, most of these studies focused on forebrain ischemic or global ischemic models that might have impeded the investigation of therapeutic HBO applications. In designing the model for their study, these investigators chose to observe and evaluate the parameters of survival time and survival rate to determine the therapeutic effect of HBO (10). Moreover, neuronal injury in the forebrain after brief global ischemia is a selective phenomenon that sometimes fully demonstrates only after observation for several days (19). By contrast, neuronal death, which follows an intense focal ischemic challenge in an area at risk for infarction, is evident within 12-24 h (19). The ischemic events characterizing neuronal death mimic more closely the clinical condition of ischemic stroke (19). Considering these conditions, we used an experimental MCAO and reperfusion model that represents a likely clinical problem: incomplete, focal ischemia with the potential for reperfusion. This model also mimics clinical disorders, such as large-vessel thrombosis, that lead to infarction, which is surrounded by ischemic penumbra. Theoretically, this model is an ideal candidate for therapeutic HBO (8).

Mainly, two patterns of cerebral damage characterize focal ischemia: focal damage and penumbral damage. Complete flow cessation causes the greatest damage at the focus. The penumbral tissues surrounding an ischemic focus sustain less severe damage because the collateral vessels supplying the penumbral area yield a residual perfusion. Oxygenation is the most critical function of blood flow; suddenly reduced oxygenation is an inevitable consequence of severe ischemia. If therapeutic intervention does not occur in a timely manner, penumbral zones could eventually lose their ability to maintain ionic homeostasis, thereby become subsumed into the focal area, and thus increasing the total ischemic brain damage. Through the administration of pharmacological agents, many investigators aim therapeutic stroke research at preventing the penumbral recruitment into the ischemic focus (6). Pharmacological intervention might either improve blood flow (i.e., oxygen supply) or protect against neuronal death. HBO treatment is considered for cerebral ischemia, because it might salvage the still viable, though nonfunctioning, tissues surrounding the infarcted area (14). Mink and Dutka. (12) reported that HBO can improve tissue oxygen delivery (especially to areas of diminished blood flow), can enhance neuronal viability, and can reduce brain edema. By raising the tissue PO₂, HBO can trigger a mechanism controlling cellular and vascular repair. HBO therapy applied after radiation injury has demonstrated increased tissue oxygen concentration, thereby stimulating angiogenesis and establishing a new capillary blood supply (5). HBO therapy salvages the still viable, though nonfunctioning, tissue surrounding the infarcted area presumably by allowing time for collateral circulation to develop. This collateralization forms the basis for the conclusion that HBO might benefit victims of cerebral ischemia. Our results showed that HBO therapy, when applied at an earlier stage after reperfusion (at 3 and at 6 h after reperfusion), decreased the infarction area in the rat MCAO and reperfusion model. Our results agree with the report of Roos et al. (16) who state that HBO therapy in the rat MCAO model without reperfusion is beneficial and agrees with the report of Krakovsky et al. (10) about other animal models of cerebral ischemia.

Despite the beneficial effects resulting from HBO therapy applied at an earlier stage (i.e., at 3 and 6 h), our results also show that HBO therapy applied at a later stage (i.e., 12 and 23 h) can yield less beneficial effects in this rat MCAO and reperfusion model. When applied at a later stage, such as 12 or 23 h after reperfusion, HBO treatment increased the infarcted cerebral area. Although the exact mechanism remains unclear, several possibilities account for the harmful effect of applying HBO therapy later. Deleterious mechanisms involved in focal ischemic injury persist throughout the postischemic period. An unresolved controversy is whether these mechanisms are 1) triggered during ischemia and persist into the postischemic period, 2) triggered during reflow (reperfusion injury), or 3) both 1 and 2 foregoing. Canevari et al. (2) reported that in a rat MCAO model (2 h occlusion), recirculation for 1-2 h temporarily restored the bioenergetic state and mitochondrial function but that secondary deterioration occurred after 4 h. Gido et al. (7) stated that delayed cell membrane dysfunction, as reflected in a rise in K⁺, occurs ~6 h after reperfusion. Despite the suggestion that the generation of free radicals during reperfusion might cause secondary damage, it remains to be determined whether HBO applied at an early stage might prevent the generation of free radicals or the damage caused by free radicals and whether HBO application at a later stage enhances the harmful effect of free radicals.

Our finding differs from the findings of other studies that demonstrate the failure of HBO to exert a beneficial effect. These differences might be related to the experimental protocols of other investigations that prescribe prolonged HBO treatment. Long-term use of HBO results in adverse effects due to the onset of oxygen toxicity as manifested by induction of lipid peroxidation and seizures (14). Long-term HBO therapy might conflict with parameters such as the hematocrit level that, in turn, might influence viscosity (14). A previous study showed that the exposure of rats to 4 ATA of oxygen for 90 min was associated with an increased level of lipid peroxidation product (malondialdehyde) and altered the enzymatic antioxidation (glutathione peroxidase) in the brain (15). Chavko et al. (4) stated that some studies used 100% O₂ at 5 ATA, which induced seizures. The Committee of the Undersea and Hyperbaric Medical Society recommends that a treatment pressure only from 2.4 to 3.0 ATA should be used at the lowest effective pressure to avoid O₂ convulsions (3).

Perspectives