l-Deprenyl Reduces Brain Damage in Rats Exposed to Transient Hypoxia-Ischemia
Background and Purpose l-Deprenyl (Selegiline) protects animal brains against toxic substances such as 1-methyl-1,2,3,6-tetrahydropyridine and 6-hydroxydopamine. Experiments were conducted to test whether l-deprenyl prevents or reduces cerebral damage in a transient hypoxia/ischemia rat model.
Methods Rats were treated for 14 days with 2 mg/kg and 10 mg/kg l-deprenyl or saline. After surgery a 20-minute hypoxia/ischemia period was induced by simultaneous occlusion of the left common carotid artery and reduction of the percentage of oxygen in the gas mixture to 10%. Rats were killed 24 hours later. Silver staining was used to reveal damage in several brain regions.
Results In the brain, both l-deprenyl dosages reduced damage up to 78% compared with the controls. Total brain damage was decreased from 23%-31% to 5%-9% with the l-deprenyl treatment (2 mg/kg: F1.13=6.956, P<.05; 10 mg/kg: F1.13=5.731, P<.05). In the striatum, significant treatment effects were found between both the l-deprenyl groups (2 mg/kg and 10 mg/kg, respectively) and the saline group (F1.13=14.870, P<.005; and F1.13=8.937, P=.01; respectively). In the thalamus, significant treatment effects were seen in the 2-mg/kg l-deprenyl group (F1.13=11.638, P<.005) and the 10-mg/kg group (F1.13=8.347, P<.05) compared with the control group. No significant damage decrease was seen in the hippocampus and the cortex.
Conclusions The results show that l-deprenyl is effective as a prophylactic treatment for brain tissue when it is administered before hypoxia/ischemia. Mechanisms responsible for the observed protection remain unclear. The regional differences in damage, however, are in accordance with the reported regional increase in superoxide dismutase and catalase activities after l-deprenyl treatment, suggesting the involvement of free radicals and scavenger enzymes.
A period of reduced oxygen supply can produce irreversible neuronal death and often causes severe disability when the major motor control areas of the brain are affected. Because of the rapid irreversibility, effort has been put into finding treatments that prevent or reduce ischemia or oligemia-induced cerebral damage.
Ischemic brain damage may involve several mechanisms, but calcium overload, glutamate toxicity, and generation of free radicals are most intensively investigated. Due to the rapid establishment of ischemic damage, pharmacological pretreatments should be considered. In our experimental hypoxia/ischemia model, several potential protective pretreatments were tested regarding their ability to reduce damage. The largest damage reduction was seen in rats that were deprived of food for 1 day despite significantly increased glutamate levels in the striatum after the hypoxia/ischemia.1
We explored l-deprenyl (Selegiline), an irreversible inhibitor of monoamine oxidase-B (MAO-b), as a potential neuroprotective agent against an ischemic/hypoxic episode. l-Deprenyl is a selective inhibitor of B-type MAO at moderate dosages and has several other characteristics that make it suitable as a prophylactic treatment in the prevention or reduction of neuronal damage.2 3 4 5 For example, l-deprenyl increases scavenger enzyme activity,3 6 and this may partially explain the described neuroprotection of l-deprenyl.4 7
In this article, the prophylactic effect of l-deprenyl on the cerebral damage of rats exposed to transient hypoxia/ischemia is described. Attention was especially focused on regional brain differences in total damage.
Materials and Methods
Animals and Treatments
Adult male Wistar rats (Centraal Proefdieren Lab), with ad libitum access to food and water and weighing 230 to 250 g at the beginning of the experiment, were divided at random into three experimental groups. These three groups were treated daily for 2 weeks with 2 mg/kg (n=8) and 10 mg/kg (n=8) IP l-deprenyl (JUMEX) or saline (n=7). Each day the animals were weighed, and the food intake was determined. Animals were treated for 14 days with 2 mg/kg l-deprenyl because this dose optimally increases the activity of the free radical scavenger enzymes.6 8 9 The high dose of 10 mg/kg l-deprenyl was used because doses larger than 2 mg/kg were shown to give neuroprotection against 6-hydroxydopamine,4 8 even after one single treatment. Blood glucose levels in the three groups were measured 14 days after initiation of the treatment (n=18) in follow-up experiments.
On day 14, rats were anesthetized with sodium pentobarbital (50 mg/kg IP), and the left femoral artery was exposed and cannulated for continuous mean blood pressure monitoring (Hewlett-Packard pressure transducer). The left carotid artery was exposed, and the rats were intubated, connected to a ventilator, and stabilized during 10 minutes with a mixture of 30% O2 and 70% N2O (infant ventilator MK2, Loosco). After stabilization, a 20-minute hypoxia/ischemia period was induced by simultaneous occlusion of the left carotid artery and reduction of the O2 content in the gas mixture to 10%, followed by a 15-minute period of normoxia (30% O2/70% N2O). During surgery, body temperature was maintained between 36.5°C and 37.5°C with an incandescent lamp and a heating pad.1
All treatments and surgical procedures were approved by a local animal ethics committee.
After a 24-hour survival period, rats were deeply anesthetized with sodium pentobarbital (70 mg/kg IP) and perfused transcardially with saline followed by 400 to 600 mL ice-cold 4% paraformaldehyde in 0.1 mol/L borate buffer (pH 9.5).10 Brains were post-fixed for 24 hours and cryoprotected overnight in 20% sucrose in 0.1 mol/L phosphate buffer, pH 7.4, at 4°C. Frontal sections 25 μm thick were cut on a cryostat microtome and stored. Every fifth section was stained with an improved silver impregnation procedure from Gallyas et al,11 12 13 which labels degenerating cell bodies and their processes. In brief, free-floating sections were rinsed three times in quartz-distilled water and pretreated for 5 minutes in a solution containing 1.1 mol/L NaOH and 75 mmol/L NH4NO3 three times. Tissue was subsequently impregnated for 10 minutes in 1.4 mol/L NaOH, 0.8 mol/L NH4NO3, and 18 mmol/L AgNO3. After being rinsed three times for 5 minutes in 47 mmol/L Na2CO3, 1.5 mmol/L NH4NO3, and 30% ethanol, the sections were developed for 1 minute in 0.07% citric acid, 0.55% formalin, 9.6% ethanol, 0.675 mol/L NaOH, and 0.75 mmol/L NH4NO3. Silver deposits were fixed for 4 minutes in 1.5 mol/L Na2S2O3·5H2O, rinsed three times for 5 minutes in distilled water, and stored in 0.1 mol/L phosphate buffer (pH 7.4) before being mounted on gelatin-coated slides.
Investigators were blinded to the procedures during surgery and analysis of the histology. Rats with a blood pressure drop during hypoxia/ischemia of less than 60% of normal blood pressure were excluded from the analysis. In frontal sections of the brain, both total and regional damage was determined with light microscopy. Camera lucida drawings of the silver staining in the side ipsilateral and contralateral to the clamped artery were made on millimeter graph paper and used for calculations of the size of the damaged area. Silver-stained area in square millimeters served as the numerator, and the total square millimeters of the area functioned as denominator. This was specified for the following brain areas: hippocampus, striatum, cortex, and thalamus. Attention was especially focused on four coronal sections: 5.7, 6.7, 7.7, and 9.2 mm rostral from the interaural line. All data shown are presented as the mean±SEM. Silver-staining data were statistically evaluated using an ANOVA with repeated measures followed by the post hoc corrected (according to Newman-Keuls) Student’s t test. Changes in weight, daily food intake, and blood pressure were analyzed with a two-tailed Student’s t test. The average silver-staining data were analyzed with ANOVA. A probability level of P<.05 was considered significant for all tests.
The Table⇓ shows that the weight gain after 14 days (Δweight) in the 10-mg l-deprenyl group (19.1±3.5 g) was markedly reduced when compared with the control group (46.9±3.1 g, P=.0001) and the 2-mg l-deprenyl group (40.3±3.0 g, P<.001), although daily food intake and food intake per gram of body weight (not shown) did not differ between the groups. Blood glucose levels in the control group were 6.3±0.3 mmol/L and were not significantly different from levels found in the 2-mg/kg and 10-mg/kg l-deprenyl–treated rats (6.0±1.0 and 6.3±0.2 mmol/L, respectively). Also, the mean arterial blood pressures before, during, and after the hypoxic period of controls and deprenyl-treated rats were not significantly different.
The percentage of damaged area when averaged over several levels showed that the forebrain in total and all regions studied, except the hippocampus and the cortex, had significantly less silver-stained surface (Fig 1⇓). In the striatum and thalamus, this damage reduction was at least 50% and at most 79%. The percentage of total damaged area in the forebrain of control rats was 27%, and this was reduced in the l-deprenyl–treated groups to 6% (2 mg/kg) and 8% (10 mg/kg).
The silver-stained surface specified at different levels rostral from the interaural line showed in some regions an anterior-posterior damage gradient. In the saline group, the brain hemisphere ipsilateral to the occluded artery was damaged for 23% to 31% of the total brain area (Fig 1A⇑). A significant reduction of this damage (by approximately 75%) to about 5% to 9% of the total brain area was seen at all four coronal levels studied in the group treated with 2 mg/kg deprenyl (F1.13=6.956, P<.05) (Fig 1A⇑). The damage in the 10-mg/kg–treated group was also significantly less (F1.13=5.731, P<.05) compared with the saline group. The Newman-Keuls test indicated that there was a significant damage difference at the most anterior (17.5±7.9%, P<.05) and posterior (15.6±3.7%, P<.05) level. The total brain damage in the other two sections, however, was not significantly different (21.8±4.9, P=.070; and 23.1±10.8, P=.071; respectively).
The localization of the silver staining in the control brain was most pronounced in the dorsolateral parts of the striatum, cortex, and thalamus (Fig 2⇓). The hippocampal area (Fig 1B⇑) in the control animals was damaged on average for 30% to 38%. In the hippocampus, no reduction of damage was seen by any of the l-deprenyl treatments.
In the saline group, silver staining was detectable in 19.6% to 24.3% of the cortical areas (Fig 1C⇑). In the l-deprenyl (2 and 10 mg/kg) groups, the cortical damage was highest at the most frontal levels (4.2% and 7.1%, respectively) but decreased gradually to 2.1% and 1.7%, respectively, at the more caudal sections. These differences were significant (P<.001) at the most caudal levels.
The caudal thalamic levels (Fig 1E⇑) of saline-treated animals showed a larger silver-stained area (50.5%) than the more frontal levels (16.7%). In the l-deprenyl–treated groups, this gradual decrease of silver staining from caudal to frontal levels was less pronounced. Both the 2- and 10-mg/kg l-deprenyl–treated groups, compared with the saline group, showed a significant neuroprotective effect in the thalamus (F1.13=11.638, P<.005; F1.13=8.347, P<.05; respectively). The thalamic damage after 2-mg/kg and 10-mg/kg deprenyl treatment was significantly reduced at the most caudal levels, at 5.7 (12.1±6.2, P<.01; and 13.6±5.9, P<.01; respectively) and 6.7 mm (9.8±3.8, P<.01; and 11.1±6.4, P<.05; respectively) rostral from the interaural line.
Of all brain areas examined, the percentage of damage was the highest in the striatal areas of the saline group (Fig 1D⇑), between 52.1% and 54.6% in the various coronal sections. In the group injected with 10 mg/kg l-deprenyl, the damage was significantly reduced (F1.13=8.937, P=.01) compared with the saline group. The Newman-Keuls test in this group showed a significant damage reduction at 9.2, 7.7, and 6.7 mm rostral from the interaural line. The group treated with the low-dose l-deprenyl showed significantly less (F1.13=14.870, P<.005) silver-stained area at all rostrocaudal levels compared with the controls and was affected on average by 21%.
This study shows that l-deprenyl exerts protective effects against ischemic/hypoxic damage in the striatum and thalamus when applied intraperitoneally to rats for 14 days in 2- and 10-mg/kg doses. No significant decrease of silver-stained areas were seen in the hippocampus and in large parts of the cortex after l-deprenyl treatment. In general, the total damaged area in both l-deprenyl groups was reduced up to 78% compared with the damage in the saline group. Neuroprotective effects of l-deprenyl also have been reported by others.14 In these experiments, l-deprenyl rescued neurons from the delayed neuronal cell death in the hippocampus14 after transient bilateral occlusion of the carotid artery.
The mechanism through which l-deprenyl diminishes damage is unclear. However, a few possibilities for the protective effects of l-deprenyl should be taken into consideration.
Previously we have shown that rats fasted for 24 hours are protected against ischemic-hypoxic damage in the presently used model.1 The reduced Δweight of the l-deprenyl–treated animals versus the saline-treated animals might suggest a chronic fasting state for the l-deprenyl–treated animals. However, the gradual gain in body weight, the unchanged food intake seen in the l-deprenyl–treated rats, and the comparable blood glucose levels are in disagreement with the idea that l-deprenyl treatment induces a hypoglycemic state in rats. This idea of a chronic fasting state is furthermore rejected by a study that has shown that the beneficial effects of l-deprenyl on life expectancy were not mediated by way of food restriction.15
The regional differences in damage protection by l-deprenyl are in agreement with MAO-b independent effects of l-deprenyl on free radical scavenger enzymes, ie, catalase and superoxide dismutase.3 6 Cortical areas showed a tendency for increased activity, but the increase in the activity of the scavenger enzymes was most pronounced in striatum and thalamus, the areas that were most protected by l-deprenyl in the present study. The hippocampal scavenger enzyme activities, however, were not changed corresponding to the lack of effect of l-deprenyl on hippocampal ischemic damage. This ability of scavenger enzymes to protect against ischemia-induced damage has been described by numerous studies. Not only exogenously applied scavenger enzymes16 but also intrinsically increased expression of these enzymes had beneficial effects on damage outcome. For instance, an increased superoxide dismutase expression in transgenic mice offered protection against radicals generated through ischemia17 18 and/or N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.19
However, other MAO-b inhibitors were shown to eliminate the rise of toxic H2O2 in the reperfusion period in a rat ischemia model.20 So it seems that l-deprenyl can prevent the damaging effects of radicals by either preventing the rise of H2O2, which can quickly be converted to the most damaging OH radical, or by increasing the enzymatic radical scavenging capacity.
Although no significant differences in protection between both doses of l-deprenyl could be found, the low dose of l-deprenyl appears to be the most effective in damage reduction. An explanation for this phenomenon might be given by the fact that l-deprenyl influences the superoxide dismutase and catalase activity in an inverted U-shaped way,3 with an optimal scavenger activity increase after administration of 2 mg/kg l-deprenyl.
Long-term l-deprenyl treatment also has several other beneficial effects: it slows down significantly the age-related decline of sexual functioning in male rats, and it proved to be beneficial for performance in a learning test.21 Finally, it also improved the lifespan of l-deprenyl–treated rats compared with their saline-treated peers.9 22
In short, a 14-day l-deprenyl pretreatment in the 2- and 10-mg/kg–treated animals offers substantial protection against an oxygen shortage. Therefore, more should be known about the exact mechanisms behind this protection, the dose responsiveness of this phenomenon, and the protective effects after prolonged l-deprenyl administration, as well as whether l-deprenyl reduces damage when given after hypoxia/ischemia.
Support for this study was obtained from the Center for Behavioral, Cognitive, and Neurosciences (BCN), University of Groningen, J.C. De Cock Foundation (grant 1193). The authors gratefully acknowledge B. Stuiver and F. Postema for technical assistance and A. Wiersma for critically reading the manuscript and statistical advice.
Reprint requests to S. Knollema, University of Groningen, Centre for Behavioral, Cognitive, and Neurosciences, Department of Biological Psychiatry, PO Box 30.001, 9700 RB Groningen, The Netherlands.
- Received September 22, 1994.
- Revision received June 26, 1995.
- Accepted June 28, 1995.
- Copyright © 1995 by American Heart Association
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