|Year : 2000 | Volume
| Issue : 3 | Page : 227--30
Effect of dichloracetate on infarct size in a primate model of focal cerebral ischaemia.
MJ Chandy, J Ravindra
Department of Neurosurgery, Christian Medical College Hospital, Vellore, 632004, India., India
M J Chandy
Department of Neurosurgery, Christian Medical College Hospital, Vellore, 632004, India.
Acidosis is a major contributing factor towards spread of the ischaemic focus in the brain. Drugs that increase pyruvate dehydrogenase activity could decrease the formation of lactic acidosis. The sodium salt of dichloracetic acid (DCA) has been found to be effective in reducing lactate. This study was undertaken to study the efficacy of DCA in reducing infarct size in experimental focal ischaemia in monkeys. Macaca radiata monkeys in the treatment group were given 35 mg per kilogram of dichloracetate intravenously immediately before occluding and interrupting the middle cerebral artery, and the control group was given saline as placebo under similar conditions. Mean infarct size expressed as a percentage of the size of the hemisphere in all the three brain slices was 35.38 in the control group as against l2.06 in the treated group (p=0. 0008).
|How to cite this article:|
Chandy M J, Ravindra J. Effect of dichloracetate on infarct size in a primate model of focal cerebral ischaemia. Neurol India 2000;48:227-30
|How to cite this URL:|
Chandy M J, Ravindra J. Effect of dichloracetate on infarct size in a primate model of focal cerebral ischaemia. Neurol India [serial online] 2000 [cited 2020 Nov 24 ];48:227-30
Available from: https://www.neurologyindia.com/text.asp?2000/48/3/227/1531
The single artery occlusion model of focal cerebral ischaemia in a non human primate by the transorbital technique without handling the brain is the closest to an ideal ischaemic stroke model. Focal cerebral ischaemia or a 'stroke' lesion consists of a central core of densely ischaemic tissue and perifocal areas of less ischaemia (penumbra). Although the central tissues are usually almost irretrievably damaged, the penumbra remains viable for longer periods depending on the collateral supply. As time progresses, there is a mismatch between blood flow and metabolic demands, inhibitory control over cell activity is temporarily offset and factors released from marginalised tissue jeopardize cells in the penumbra.,,, Surrounding perifocal tissue may be salvaged by reperfusion or addition of pharmacological agents which prevent this self destruction. The main factors which jeopardize cell survival in the ischaemic penumbra are acidosis, oedema, transient K+ efflux/Ca++ influx and depressed protein synthesis., Under aerobic conditions glucose is at first broken down to pyruvate, resulting in 2 mol of ATP, and in the presence of oxygen pyruvate is then metabolised, by pyruvate dehydrogenase and by a series of mitochondrial reactions, to CO2 with the formation of 36 mol of ATP. Hypoxia retards these events and leads to the reduction of pyruvate to lactate. Although this reaction does not produce H+, anaerobic metabolism leads to the production of lactate and hydrogen ions. The energy disadvantage of the aerobic anaerobic transition is that ATP production is reduced, mitochondria deprived of oxygen do not sequester calcium and the cell is acidified. It is well known that acidosis predisposes to the spread of the ischaemic focus.,,,
Treatment of lactic acidosis is traditionally by the administration of base, although clinically this has several drawbacks. Drugs that increase pyruvate dehydrogenase activity are said to decrease formation of lactic acid and acidosis. The sodium salt of dichloracetic acid is one such drug.
Sixteen adult Macaca monkeys of either sex with weights between 5 to 10 lb were chosen for the study. The animals were divided into two groups. Initially the study was done without blinding the investigator and this included four monkeys in the treatment group with DCA and four for controls with placebo (Part 1). The second group of four treated and four control animals were done as a blinded placebo controlled study to eliminate operator bias (Part 2). The monkeys were anaesthetised using intravenous pentabarbitone at a dose of 25 mgm per kilogram. Anaesthesia was maintained with intermittent doses of pentabarbitone as required. The monkeys were monitored carefully while blood gases and mean arterial pressure were maintained at a steady state during the experiment. The sodium salt of DCA was freshly prepared by neutralising the acid (Fischer scientific company) with sodium bicarbonate and buffered to pH of 7.2 with standard phosphate buffer. A solution of the salt was prepared to a strength of 35mgm per ml. In part 2 of the study, DCA and a similar solution of saline was identically packed in the pharmacy and blinded.
The transorbital technique was used to expose the intracranial structures without handling the brain. The middle cerebral artery (MCA) was exposed through the orbit. After exposing the internal carotid, its bifurcation, the middle cerebral artery and its perforators, the MCA was coagulated and cut proximal to the perforators. The wound was closed, orbit packed with dental cement and the animals returned to their cage. After 24 hours the monkeys were reanaesthetised, a quick craniotomy done and brain removed after sectioning the optic nerves and brain stem at the level of the tentorium. Standard coronal sections of the brain were made at the level of the temporal pole, optic chiasma and the mamillary body. The brain slices were then stained using the following technique. The brain slices were completely immersed in a 1% solution of 2,3,5-triphenyl tetrazolium chloride (TTC) in saline with a pH of 7.2 and incubated at 37oC for ten minutes in the dark as TTC is a light sensitive chemical. After fifteen minutes the reaction was arrested using 10% buffered formalin which was also used to fix the stain. Normal areas stain red whereas infarcted areas remain pale. The macroscopic infarct size was quantified using a plastic grid, each square measuring four square millimeters. The posterior surfaces of each of the three slices were stained and the infarct size expressed as a percentage of the total surface area of the hemisphere. The infarct sizes in both Part 1 and Part 2 of the experiment were compared to check for any variation between the results in these two groups. Using the Wilcoxin and the Mann-Whitney two sample test, it was seen that there was no statistically significant difference between the controls (p=0.08326 ) and the treated groups (p=0.38647).
Part 1 : In this group of four monkeys used as controls, an infusion of 100 ml of saline was given just prior to coagulating and cutting the middle cerebral artery. The tabulated results show that in the control group there was mean infarct size of 37.6 % with a standard deviation of 3.52 [Table I] and [Table II]. In the treated group of four monkeys who were given DCA in 100 ml of saline, the mean infarct size was 12.95% with a standard deviation of 1.83 [Table I] and [Table II]. Part 2 : In this part of the experiment, the control and DCA solutions were packed similarly in the pharmacy with a standard deviation of 3.47 [Table I] and that of the treated group was 11.17% with a standard deviation of 2.61 [Table II]. The percentage of area of infarction between the study and the control groups in all the three slices were compared as a whole and it was seen that the maximum reduction in the infarct was seen in the second slice corresponding to the insular and medial temporal cortices. The percentage of infarction was compared as a whole between the control and study groups and was found to be 35.38% in the control group and 12.06% in the study group [Table III], [Table IV] and [Table V]. These values were found to be highly significant (p=.0008).
The fact that focal cerebral ischaemia has a central zone of ischaemic necrosis with irreversible damage, surrounded by an area of borderline ischaemia which is potentially reversible, has led workers to study various agents that may reduce the ischaemic penumbra, thus reducing the eventual damage caused by the ischaemic process. Following cerebral ischaemia there is an accumulation of lactic acid which is a major cause of accentuated tissue injury resulting in cell death. It has been shown that DCA causes a reduction in tissue and serum lactate in experimental cerebral ischaemia. This study was designed to evaluate the beneficial effect of DCA in reducing infarct size in experimental focal ischaemia in monkeys. The lactate lowering effect of DCA is mediated by the stimulation of the activity of the enzyme pyruvate dehydrogenase (PDH) which limits the rate limiting step in the aerobic oxidation of pyruvate and lactate, thus enhancing the aerobic oxidation of glucose resulting in a better supply of high energy phosphate bonds., Sodium bicarbonate, which has been traditionally used in the treatment of lactic acidosis, has been found to be inadequate in the treatment of ischaemic lactic acidosis. Katayama and Welsh in their experimental study of ischaemia and reperfusion in gerbils have shown that the therapeutic effect of DCA lies in the prevention of secondary energy failure caused at the time of recovery of neuronal tissue from the ischaemic insult. At this stage, the metabolic demand for high energy phosphate is increased and by activating PDH and meeting this demand, DCA prevents energy failure. Other workers have shown in their study of global forebrain ischaemia in rats that increase in lactates occur after about ten minutes after the onset of ischaemia., It is also known that the peak levels of DCA, after its infusion, occur within the hour. Although it is well known that numerous factors play a role in the development and progression of the ischaemia, it was not known if control of the lactic acidosis alone would reduce the infarct size. In the present study it was shown that pretreatment with DCA resulted in a significant reduction of infarct size under controlled conditions. The maximal decrease in the infarct size was seen in slice 2 going through the optic chiasm which corresponds to the insular and the mesial temporal cortices which receive collateral circulation from both anterior and posterior cerebral arteries. These are the areas that correspond to the ischaemic penumbra where lactic acidosis play a significant role in determining the degree of residual infarcted tissue. In the present study it has been noted that pretreatment with the sodium salt of dichloracetic acid resulted in a significant reduction of residual brain infarction in an experimental monkey model of focal cerebral ischaemia. This finding would have particular relevance with regard to brain protection during temporary clipping of major arteries during cerebral vascular surgical procedures.,
|1||Diaz FG, Ausman JI : Experimental cerebral ischaemia. Neurosurgery1980; 6 : 436-445. |
|2||Chopp M, Frinak S, Walton DR et al : Intracellular acidosis during and after cerebral ischaemia. In vivo nuclear magnetic resonance study of hyperglycaemia in cats. Stroke1987; 18 : 919-923. |
|3||Katayama Y, Welch FA : Effect of dichloracetate in regional energy metabolites and pyruvate dehydrogenase activity during ischaemia and reperfusion in gerbil brain. J NeuroChem 1989; 52 : 1817-1821. |
|4||Bederson JB, Pitts LH, Germana SM et al : Evaluation of 2,3,5- triphenyl tetrazolium salt as a stain for detection and quantification of experimental cerebral infarction in rats. J Cereb Blood Flow Metab 1978; 2 : 210-215. |
|5||Rehncrona S, Rosen I, Seisjo BK : Brain lactate acidosis and ischaemic cell damage. 1. Biochemistry and Neurophysiology. J Cereb Blood Flow Metab 1981; 1 : 297-311. |
|6||Combs ET : Relationship between plasma glucose, brain lactate and intracellular pH during cerebral ischaemia in gerbils. Stroke1990; 21 : 936-942. |
|7||Garcia JH : Experimental ischaemic stroke : A review. Stroke 1984; 15 : 5-14. |
|8||Kalimo H, Rehncrona S, Soderfelt B et al : Brain lactic acidosis and ischaemia cell damage. 2. Histopathology. J Cereb Blood Flow Metab 1981; 1 : 313-327. |
|9||Waters WC III, Hall JD, Schwartz WB : Spontaneous lactic acidosis: the nature of the acid base disturbance and considerations in diagnosis and management. Am J Med 1963; 35 : 781-793. |
|10||Wells PG, Moore GW, Stacpoole PW : Metabolic effects and pharmocokinetics of intravenously administered dichloracetate in humans. Diabetologica1980; 19 : 109-113. |
|11||Stacpoole PW : Treatment of lactic acidosis with dichloracetate. N Eng J Med 1981; 309 : 390-396. |
|12||Romeh SA, Tannen RL : Therapeutic benefits of dichloracetate in experimentally induced hypoxic lactic acidosis. J Lab Clin Med 1986; 107 : 378-383. |
|13||Fredrichs KU, Lindsberg PJ : Increased cerebral lactate output to cerebral venous blood after forebrain ischaemia in rats. Stroke1990; 21 : 614-617. |
|14||Krain RP, Pulsinelli WA, Plum F : Carbonic acid buffer changes during complete brain ischaemia. Am J Physiol 1986; 250 : 348-357. |
|15||Plum F : What causes infarction in ischaemic brain? The Robert Wartenburg Lecture. Neurology 1983; 33 : 222-233. |
|16||Ranjan A, Theodore D, Haran RP et al : Ascorbic acid and focal cerebral ischaemia in a primate model. Acta Neurochir (Wien)1993; 123 : 87-91. |
|17||Henry PT, Chandy MJ : Effect of ascorbic acid on infarct size in experimental focal cerebral ischaemia and reperfusion in a primate model. Acta Neurochir (Wien)1998; 140 : 9 : 977-980. |