reperfusion insult

Experimental Neurology 200 (2006) 290 – 300 www.elsevier.com/locate/yexnr

Reduction in oxidative stress and cell death explains hypothyroidism induced neuroprotection subsequent to ischemia/reperfusion insult Leena Rastogi a , Madan M. Godbole a,⁎, Madhur Ray d , Priyanka Rathore d , Sunil Pradhan b , Sushil K. Gupta a , Chandra M. Pandey c a

Department of Endocrinology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareli Road, Lucknow-226014, India b Department of Neurology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareli Road, Lucknow-226014, India c Department of Biostatistics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareli Road, Lucknow-226014, India d Division of Pharmacology, Central Drug Research Institute, Mahatma Gandhi Marg, Lucknow-226001, India Received 21 September 2005; revised 4 February 2006; accepted 10 February 2006 Available online 17 April 2006

Abstract Hypometabolic state following hypothermia is known to protect tissues from ischemic injury. Hypothyroidism produces a hypometabolic state. The present study was undertaken to investigate the protective effects of hypothyroidism following cerebral ischemia and to ascertain the underlying mechanism. Euthyroid (E) and hypothyroid (H) animals were exposed to a 2 h of middle cerebral artery occlusion followed by 24 h of reperfusion (I/R). Specific enzymatic methods and flowcytometry were used to assess the quantitative changes of molecules involved in neuronal damage as well as in protection. As compared to euthyroid ischemic reperfused (E + I/R) rats, H + I/R rats had insignificant neurological deficit, and smaller area of infarct. H + I/R rats had significantly lower markers of oxidative stress, and lactate dehydrogenase (LDH) activity (a marker for necrosis). Natural antioxidant activity (particularly superoxide dismutase) and integrity of mitochondria (membrane potential) were maintained in H + I/R group but not in E + I/R group. The number of neurons undergoing apoptosis significantly lower in hypothyroid ischemic rats as compared to euthyroid ones. These results suggest that hypothyroid animals face ischemia and reperfusion much better compared to euthyroid animals. A possible explanation could be the decreased oxidative stress and maintained antioxidant activity that finally leads to a decrease in necrosis and apoptosis. These observations may suggest strategies to induce brain-specific downregulation of metabolism that may have implications in the management of strokes in human beings. © 2006 Elsevier Inc. All rights reserved. Keywords: Stroke; Hypothyroidism; Oxidative stress; Necrosis; Apoptosis

Introduction

Abbreviations: E, euthyroid; H, hypothyroid; I/R, ischemia/reperfusion; E + I/R, euthyroid plus ischemia/reperfusion; H + I/R, hypothyroid plus ischemia/ reperfusion; LDH, lactate dehydrogenase; TH, thyroid hormone; BMR, basal metabolic rate; T3, triiodothyronine; MMZ, 2-mercapto-1-methylimidazole; MCA, middle cerebral artery; TTC, 2,3,5-triphenyltetrazolium chloride; H&E, haematoxylin and eosin; TUNEL, transferase dUTP nick end labeling; NADPH, reduced pyridine nucleaotide; ROS, reactive oxygen species; Ca2+, intracellular calcium; SOD, superoxide dismutase; MPO, myeloperoxidase; MDA, melondialdehyde; H2O2, hydrogen peroxide; FITC, fluorescien isothiocynate; PI, propidium iodide. ⁎ Corresponding author. Fax: +91 522 2668017, 2668973. E-mail addresses: [email protected], [email protected] (M.M. Godbole). 0014-4886/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2006.02.013

Stroke is the third leading cause of death in developed countries. About 25% of sufferers die due to stroke or its complications while 50% have moderate to severe long-term neurological disabilities. Only 26% recover most or all normal health and function (Reed et al., 1999). Though there have been several efforts focussing on the vascular front to reduce infract size or neurological disabilities in human beings in the initial 3–6 h after stroke modalities to render brain tissue less prone for ischemic damage (i.e., neuroprotection) are still in the experimental stage. Several mechanisms of neuronal injury in stroke have been proposed. These include release of excitatory amino acids, calcium overload, protein inhibition and

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formation of free radicals (Kristian and Siesjo, 1998). It is well known that ischemia is a condition where tissues experience less oxygen and substrate availability. Hypothyroidism/ hypothyroxinemia also appear to be protective under certain physiological/pathological situations such as in the last trimester of pregnancy (to protect the fetal brain) in euthyroid sick syndrome (as a consequence of low metabolic rate) and during cardiac transplant (to ensure better recovery) (Chopra, 1996; Galinames et al., 1994; Sinha et al., 1997). Decreased glutamate release following stroke in hypothyroid gerbils provided the preliminary experimental evidence to favor this hypothesis (Shuaib et al., 1993, 1994). In vivo, thyroid hormone (TH) is the key hormone involved in setting the basal metabolic rate (BMR) on a long-term basis in target tissues such as liver, heart, kidney and brain. Hypothyroidism results in generalized decrease in BMR, decreased energy expenditure, oxygen requirement and reduced substrates utilization (Sterling et al., 1977). Several authors have shown protective effect of hypothyroidism against free radical induced damage in kidney, liver and lung tissue (Paller and Sikova, 1986; Swaroop and Ramasarma, 1985; Yam and Roberts, 1979). Brain, unlike other organs of the body, is normally thought to be well protected from peripheral effects of hypothyroidism. The availability of T3 to the brain is a function of high Km type II deiodinase; it requires very little thyroxine which crosses through blood brain barrier. The understanding of the mechanism through which hypothyroidism may protect the brain following ischemia–reperfusion injury is essential for identification of targets that can be specifically addressed in early post ischemic conditions. The present study was undertaken to investigate the effect of hypothyroidism on oxidative stress and neuronal cell death in cerebral cortex following focal ischemia. Materials and methods Animals and experimental protocol One hundred and thirty male Sprague–Dawley albino rats (weight 250–350 g) were housed at a temperature of 25 ± 2°C with alternating 12 h light and dark cycles and free access to standard food pellets and water. The rats were divided into two groups: euthyroid (E, n = 60) and hypothyroid (H; n = 70). The rats were rendered hypothyroid by administration of 2-mercapto-1-methylimidazole (MMZ) in their drinking water at a concentration of 0.025% (w/v) for first 3 weeks and 0.01% (w/v) in the subsequent 3 weeks. After a period of 6 weeks, levels of TH were measured by radioimmunoassay using kits from Diagnostic Product Company (New York, USA) in blood samples drawn from animal tail. Sham operated E and H group rats retained the same nomenclature and those who underwent ischemia/reperfusion were labeled as E + I/R and H + I/R group, respectively. Body weights of all the animals were monitored at weekly intervals as well as at the beginning of occlusion/reperfusion. The femoral artery was cannulated to record blood pressure and heart rate through a Statham P23 DC pressure transducer and P44 tacograph,

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respectively, on different channels of a Grass model 7 polygraph, (Grass Instruments Co., Quincy, Mass, USA). Body temperature was monitored in each group of rats before surgery, at the beginning of reperfusion and at the time of sacrifice. It was recorded manually by thermo-probe temporarily inserted 2 cm into the rectum. Transient focal ischemia Rats were fasted overnight and anaesthetized by an intraperitoneal injection of 30 mg/kg sodium pentobarbital. Right middle cerebral artery (MCA) was occluded by advancing a 4.0 nylon filament from right external carotid up to middle cerebral artery for 2 h (Zea-Longa et al., 1989) and retracting the same gently to allow the reperfusion for next 24 h. In equal number of euthyroid and hypothyroid rats the sham operation was performed. Body temperature was maintained 36–38°C by using a heating lamp. Blood collection and tissue sampling Rats were anesthetized and blood was collected both from the right jugular vein (ipsilateral side) and cardiac puncture for enzymatic assays. While 20 rats from each group were sacrificed at 24 h after Ischemia/reperfusion and sham operation (Total 80 rats), 10 rats from each groups were sacrificed at 72 h after Ischemia/reperfusion and sham operation to perform the 2,3,5-triphenyltetrazolium chloride (TTC) staining and estimate LDH activity and lipid peroxidation to assess the effect of injury at a longer time point (Total 40 rats). From each group of 20 rats, brain tissue from both the hemispheres were stained after dissection and used for TTC staining (n = 5). Paraformaldehyde perfusion was performed prior to dissection and tissue collection for haematoxylin and eosin (H&E) staining and transferase dUTP nick-end labeling (TUNEL), (n = 5). Only the right hemisphere tissue was used from the remaining 10 rats for carrying out biochemical and cellular estimations after confirmation of the infarct size in a few coronal sections from both hemispheres. A part of fresh tissue portion was used for isolation and purification of neuronal and mitochondrial preparations. Purified neuronal preparations were used to assess apoptotic cell death and mitochondrial preparations were used for the estimation of reduced pyridine nucleotide (NADPH), reactive oxygen species (ROS), intracellular calcium (Ca2+) and membrane potential. A portion of right hemisphere from all the 10 rats was stored at −70°C for estimating superoxide dismutase (SOD), catalase, myeloperoxidase (MPO), LDH activity and malondialdehyde (MDA) levels. An additional set of experiment was performed to assess the effect of even mildly purported hypothermia in hypothyroid rats subsequent to ischemia/reperfusion injury. Hypothyroid ischemic rats (n = 10) were divided into two groups immediately after the start of reperfusion; in one group, body temperature of 37°C was maintained by keeping the rats under a heating lamp for 24 h while the other group of rats were kept initially warm till the time the anaesthesia effect wore out (2 h) and then

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keeping them at ambient temperature of 25°C for next 22 h. Infarct assessment by TTC and measurements of MDA and LDH activity were performed. Power of test was applied to validate the adequacy of number of animals used for each experiment and estimation. All animal procedures performed were in strict accordance with the institutional guidelines for animal care and research. Neurological evaluation All the animals were subjected to neurological evaluation by using a 6-point postural reflex test (Bederson et al., 1986). Briefly, scoring was as follows, 0 = no deficit, 1 = failure to extend left forepaw, 2 = circling to the left while pulled by tail, 3 = paresis of the left side, 4 = No spontaneous walking and 5 = death. Infarct assessment and quantitation For TTC staining, the fore brain was quickly removed and sliced into 2 mm thick coronal sections. Seven coronal brain sections were cut from freshly obtained cleaned tissue and stained with TTC. All the slices were incubated for 30 min in 2% solution of TTC at 37°C and fixed in 10% paraformaldehyde solution. The area of infarction of each section was determined using a computerized image analysis system (software from Biovis image plus, India). Total lesion volume was calculated by summing up the infarct areas in each section and multiplying it by slice thickness. Histopathology (H&E) and TUNEL staining Rats were perfused with 100 ml of saline and 200 ml of 4% paraformaldehyde in phosphate buffered saline (pH 7.4). Subsequently, the brain was placed in the fixative overnight and was embedded in paraffin. Coronal sections of the brain were cut from 1 mm behind the bregma, and stained with H&E. Transferase dUTP nick-end labeling (TUNEL) staining TUNEL staining was performed with ApoAlert DNA Fragmentation Assay Kit (CLONTECH Laboratories, Inc. USA) in paraffin embedded tissue sections according to the manufacturer's instruction. This fluorescence assay kit detects apoptosis induced nuclear fragmentation. In brief, tissue sections were deparaffinized in xylene and hydrated in a sequence of ethanol washing, followed by a final wash in phosphate buffer saline (PBS). Nuclei of tissue sections were stripped off protein by incubation with 20 μg/ml proteinase K for 15 min. The slices were then washed in distilled water and PBS and incubated in a premix containing equilibration buffer, nucleotide mix and TdT enzyme for 1 h at 37°C. The reaction is terminated with 2× SSC (0.3 M sodium chloride and 30 mM sodium citrate buffer). Slides were counterstained with Hoechst dye 33258 (1 μg/ml) solution. Slides were viewed under microscope using appropriate exciter and barrier filters.

Tissue processing and measurement of enzymatic activity Tissue samples stored at −70°C were homogenized individually after slowly bringing to room temperature. Ten percent (w/v) brain homogenate in ice cold phosphate buffer (50 mM, pH 7.0) was prepared using an ice-cold Teflon homogenizer at 2000 rpm by three 10-s up and down strokes over a 3-min period. Two-ml aliquot of homogenate was used for myeloperoxidase (MPO) activity measurement. Rest of the homogenate was centrifuged at 2000 rpm for 10 min at 4°C. In the supernatant levels of malondialdehyde (MDA) and activities of lactate dehydrogenase, superoxide dismutase (SOD) and catalase were determined. Measurement of Lipid Peroxidation MDA was determined by quantifying the reaction product with thiobarbituric acid in tissue homogenate (Okhawa et al., 1979). The colored end product was read at 540 nm. Results were expressed as nmoles MDA/mg protein. Myeloperoxidase activity MPO activity was measured in brain homogenate containing 0.5% hexadecyltrimethylammonium bromide. The samples were freeze–thawed and sonicated three times (10 s each). Samples were incubated at 4°C for 20 min and centrifuged at 16,000 × g for 15 min. MPO activity was measured spectrophotometrically in the supernatant using 0.53 nM o-dianisidine dihydrochloride, 0.15 nM H2O2 (Barone et al., 1991). One unit of MPO activity was defined as that degrading 1 μmol of H2O2 in 1 min. Results were expressed as U/min/g wet tissue. Determination of superoxide dismutase SOD activity was measured in supernatant (Kakkar et al., 1984). SOD activity was expressed in terms of U/mg protein, where 1 Unit is defined as that amount of enzyme that inhibits the optical density at 560 nm of chromogen production by 50% under the above assay conditions. Results were expressed as U/min/mg protein. Determination of catalase Catalase activity in the supernatant was measured by recording the rate of decrease in H2O2 absorbance at 240 nm (Aebi, 1974). The activity of catalase is expressed as μmol H2O2/min/mg protein. Assessment of the extent of apoptosis/necrosis in purified neuronal preparations Homogenous suspension of individual neurons was prepared in HEPES–Tyrode solution (145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose, 5 mM HEPES, pH 7.4) by mild treatment of the finely chopped forebrain tissue with

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dispase (1000 protease unit/ml) for 45 min at 37°C. After enzymatic treatment the dissociated neurons were passed through a filter (∼53 μm) to remove large neurons and tissue fragments (Oyama et al., 1996; Sureda et al., 1996). Dissociated neurons (homogenous with viability of 85– 95%) were loaded with propidium iodide and flourescein isothiocynate (FITC). Early apoptosis in neurons was measured using Annexin V-FITC apoptosis kit (Oncogene Research Product, USA) following instructions given by the manufacturer for flow cytometry. This Annexin V-FITC apoptosis kit uses annexin V conjugated with FITC to label phosphotidylserine sites on the membrane surface. The kit includes Propidium iodide (PI) to label the cellular DNA in necrotic cells where the cell membrane has been totally compromised. This combination allows the differentiation among early apoptotic cells (annexin V positive, PI negative), necrotic cells (annexin V positive and PI positive), and viable cells (annexin V negative, PI negative). FITC and PI signals are detected at 518 nm and 620 nm using FL1 and FL2 detectors, respectively. The log of Annexin V-FITC fluorescence is displayed on the x axis and the log of PI fluorescence, on the y axis. Data were analyzed using Cell Quest Software (Becton Dickinson USA). For each sample, 10,000 cells (events) were acquired. The flowcytometry data was expressed as mean ± SD from three independent experiments. Measurement of lactate dehydrogenase activity in tissue homogenate and plasma and nitrite content in plasma LDH activity in tissue homogenate and in plasma was estimated with the reagent kit (Boehringer Mannheim, Germany), based on the oxidation of lactate to pyruvate with simultaneous reduction of NAD to NADH. The rate of NAD reduction was measured as an increase in absorbance at 340 nm. Results were expressed as U/min/mg protein. Nitrite levels were estimated by using Greiss reagent (Singh, 1997). Equal volumes of reagent (2% sulphanilamide in 5% H3PO4 and 0.2% n-Clnapthyl-ethylenediamine dihydrochloride) and plasma (1:1) were incubated for 15 min at 37°C and absorption was measured at 540 nm. Results were expressed as nmole NO2/mg plasma protein. Measurement of reactive oxygen species, intracellular calcium, membrane potential and energy level in isolated mitochondria

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and 6 mM succinate) and centrifuged at 17,000 × g for 5 min. The pellet was finally suspended in the same medium. Flow Cytometer assessed mitochondrial viability using propidium iodide. Intracellular calcium content The suspended mitochondrial pellet (0.3 mg/ml) were loaded with Fura-2 AM (1 μmol/l) for 30 min at 37°C and then washed twice in sodium succinate medium. Fura-2 fluorescence was read in a spectrofluorophotometer (Shimadzu RF-5000) at dual excitation of 340 and 380 nm and emission at 510 nm. Alterations in the calcium levels were monitored by the change in 340/380 ratio (Zaiden and Sims, 1994). Reactive oxygen species The suspended mitochondrial pellet (0.3 mg/ml) was incubated with 50 μM 2′7′-dichlorofluorescin diacetate (DCFH-DA) for 30 min at 37°C, washed twice in succinate medium and fluorescence was monitored at excitation 488 nm and emission 530 nm (Guanasekhar et al., 1995). Mitochondrial membrane potential Mitochondria (0.3 mg/ml) in succinate medium were incubated with Rhodamine 123 (1 μg/ml) for 30 min at 37°C. The mitochondria were washed twice and resuspended in buffer, before measuring changes in membrane potential by flow cytometer at an excitation of 488 nm and emission of 530 nm (Emaus et al., 1986). NADPH level estimation As reduced Pyridine nucleotide (NADPH) shows fluorescence and NAD is nonfluorescent, excitation at 340 nm and an emission at 450 nm were used to obtain NADPH fluorescence in suspended mitochondrial pellet (0.3 mg/ml) without using any reagent (Chance and Balcheffsky, 1958). Protein assay Protein was assayed in all tissue homogenate, mitochondrial preparation and in plasma utilizing bovine serum albumin as an external standard to express specific enzyme activity (Lowry et al., 1951). Statistical analysis

Mitochondria were isolated as per the described procedure (Lai and Clark, 1990). The tissue was homogenized (10% w/v) in ice-cold buffer containing 0.32 M sucrose, 1 mM EDTA and 10 mM Tris–HCl, pH 7.4 and centrifuged at 1800 × g for 10 min. The pellet homogenized and centrifuged at 1800 × g for 10 min. The supernatant from the first and second spins were added together and centrifuged at 17,000 × g for 20 min. The pellet was resuspended in sodium succinate medium (250 mM sucrose, 5 mM KH2PO4

Results of quantitative parameters were presented as mean ± Standard deviation. Statistical comparison were made using Student's t test for comparison between two groups and one way analysis of variance for more than two groups. To find out group differences Post Hoc analysis of variance was done using Student–Newman–Keul's (SNK) test. Data were analyzed by statistical software SPSS. P value <0.05 was considered significant.

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Results Thyroid hormone status and physiological parameters Rats tolerated the experimental protocol well and no deaths occurred in any group. Circulating thyroid hormone levels confirmed that MMZ treatment had produced a thyroid hormone deficient state. In MMZ-treated rats serum T3 and T4 levels were reduced to 40% (P < 0.05) and 85% (P < 0.05) from the euthyroid controls, respectively (Table 1). Hypothyroid rats showed significant reduction in blood pressure, heart rate and body weight in comparison to euthyroid rats (P < 0.05). The mean body temperature of euthyroid and hypothyroid group of rats was 37 ± 0.2°C and 35.9 ± 0.2°C, respectively, before occlusion and 37.3 ± 0.1°C and 36 ± 0.2°C before the beginning of reperfusion. The rats usually recovered from anaesthesia approximately 2 h after the surgical procedure. We performed additional experiments to assess the effect of even mildly purported hypothermia in hypothyroid rats subsequent to ischemia/reperfusion injury. No significant changes were observed in injury parameters namely infarct size, LDH activity and MDA levels in H + I/R rats kept in a warm light device, as compared to H + I/R rats kept at a room temperature of 25°C (Data not shown). Postural reflex (neurological deficit) All rats showed normal postural reflex (score = 0) before intervention. The lower score is indicative of better neurological performance. After 24 h of MCA occlusion/reperfusion, H + I/R group showed no (15 rats) or minimal (5 rats) neurological deficit while E + I/R group showed moderate deficit of 2.0 ± 0.7 grade. In H + I/R group, rats with no neurological deficit had T3 levels of 0.86 ± 0.2 nM as compared to rats with mild neurological deficit (score 0.8 ± 0.2; serum T3 levels of 1 ± 0.2 nmol/l, P < 0.05). At 72 h, E + I/R group rats showed neurological deficit (score 3.0 + 0.4), while no such increase was observed in H + I/R group rats (score <1.0). Infarct assessment Infarct assessment by 2,3,5-trimethyl tetrazolium chloride (TTC) staining Out of 20 hypothyroid rats that underwent Ischemia/ reperfusion insults, 15 showed no detectable infarct while 5

rats showed some morphological damage at 24 h. The representative picture of TTC stained cerebral hemisphere indicating partial infarct is shown in Fig. 1. The infarct size in right hemisphere in E + I/R group was significantly larger (46%) as compared to H + I/R group (10%). Similarly, infarct volume was significantly larger in E + I/R group (270 ± 32.5 mm3) as compared to H + I/R group (98.46 ± 20.6 mm3; P < 0.05). At 72 h, infarct volume in E + I/R group and H + I/R group was 280 ± 39.0 mm3 and 100 ± 25.5 mm3, respectively (P < 0.05) with no further deterioration in H + I/R (Fig. 1). Infarct assessment by Haematoxylin and Eosin (H&E) Infarct histology was assessed by H&E stain in euthyroid and hypothyroid control and as well as their respective ischemia/reperfusion group rats (Fig. 2, panel A). Histological examination of brain at 24 h in E+ I/R group showed a consistent pattern of ischemic brain damage. There were less ischemic neurons in H + I/R rats in comparison to E + I/R rats. Euthyroid and hypothyroid control rat brain showed normal morphology of neurons. Hypothyroidism prevents apoptotic cell death subsequent to ischemia/reperfusion Fluorescence microscopic detection of fragmented DNA incorporating fluorescein-dUTP revealed almost absence of apoptotic cells in euthyroid control brain. The numbers of cells exhibiting TUNEL fluorescence were maximal in euthyroid ischemic reperfused rat brain. Though the number of cells that underwent apoptosis under hypothyroid condition was higher compared to euthyroid controls it remained unaltered subsequent to ischemia/reperfusion injury (Fig. 2, panel B). Further, apoptosis was quantified in neuronal rich cell preparations using fluorescence activated cell sorting analysis. A representative plot is depicted in Fig. 2, panel D. The data using annexin V labeling indicated the near absence of apoptosis in euthyroid control rat neuronal preparations. On ischemia/reperfusion, significantly large number of neurons (34 ± 5%) in euthyroid rats underwent apoptotic cell death compared to any other group of rats (P < 0.05). Though the extent of apoptotic neurons is significantly higher in hypothyroid control rat neurons (12.84 ± 2.5%) compared to euthyroid control rat neurons, no significant increase was observed on reperfusion of hypothyroid ischemic rats (16.38 ± 1.5%).

Table 1 Physiological variables in both groups of rats Groups

Euthyroid Hypothyroid

Body weight (g)

Blood pressure (mm mercury)

Heart rate (Beats/min)

Thyroid hormone

Body temperature in °C

T3 (nM)

T4 (nM)

Before surgery

At time surgery

After reperfusion

270 ± 30 220 ± 10*

106 ± 10 85 ± 5*

280 ± 20 240 ± 10*

1.35 ± 0.05 0.87 ± 0.02*

56.01 ± 10 8.03 ± 1.03*

37 ± 0.2 35.9 ± 0.2*

37.3 ± 0.1 36 ± 0.2*

37.1 ± 0.1 36 ± 0.5

Blood pressure and heart rate were measured in 5 animals in each group. Other parameters were measured in 20 animals in each group. Data were expressed as mean ± SD and statistical significance was assessed by Student's t test. *P < 0.05 when comparison were made between euthyroid and hypothyroid rat.

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Fig. 1. TTC stained coronal section of different group of rat brain. Representative photograph of one animal from euthyroid control (a), hypothyroid control (d); euthyroid (b and c) rat and hypothyroid (e and f) rat after 2 h of transient MCA occlusion and animals were sacrificed after 24 and 72 h of reperfusion, respectively. 2-mm thick brain sections were stained with TTC. The unstained areas represent the infarcted brain tissue. Pre-treatment with MMZ decreased infraction significantly in the 24 and 72 h of reperfusion in brain (P < 0.05). Infarct volume in panels (b and e) group was 270 ± 32.5 mm3 and 98.46 ± 20.6 mm3, respectively. Infarct volume in panels (c and f) group was 280 ± 39 mm3 and 100 ± 25.5 mm3, respectively. Number of animals used for infarct volume was five in each group.

Fig. 2. Effect of hypothyroidism on haematoxylin–eosin staining and TUNEL in paraffin embedded brain section and extent of apoptosis and necrosis in isolated neurons from different group of rats (Annexin V-FITC). Histological photomicrographs of brain sections of different group of rats. Panel A shows haematoxylin and eosin staining, original magnification ×100. Panel B shows TUNEL staining, original magnification ×10. Bright green fluorescence shows TUNEL-positive cells. Panel C shows Hoechst 33258 nuclear staining. Panel D shows extent of apoptosis and necrosis in neurons by flow cytometry (Cell Quest Analysis system) with Annexin V and propidium iodide. The cytogram in panel D represents percentage of cells; lower left hand quadrant-viable cells, lower right hand quadrant-early apoptosis, Upper right hand quadrant-late apoptotic/necrotic and upper left hand quadrant-dead cells. Different groups are denoted as euthyroid control (E), euthyroid ischemic reperfused rats after 24 h (E + I/R), hypothyroid control (H), and hypothyroid ischemic reperfused rat after 24 h (H + I/R).

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Tissue homogenate and plasma

Lactate dehydrogenase activity

Superoxide dismutase and catalase activity Significant differences were observed in SOD activity among groups. On post hoc analysis, it was observed that significant reduction in SOD activity was primarily due to Ischemia/reperfusion in euthyroid rats. On the other hand, SNK post hoc analysis showed that euthyroid control, hypothyroid control and H + I/R rats fall in the same homogenous subset (Table 3). The catalase activity in brain remained unchanged in all the groups (Table 3).

LDH activity, an index of tissue injury was estimated in plasma and tissue homogenate of all groups of animals. LDH activity was comparable in euthyroid and hypothyroid rats before occlusion. A many fold increase in the activity of LDH in plasma and tissue was observed in euthyroid ischemic reperfused rats (E + I/R) in comparison to euthyroid control rats (P < 0.001). A significant (67%) decrease in LDH activity was observed in H + I/R group in comparison to E + I/R after 24 h (P < 0.001) (Table 2). Amelioration of tissue injury effect by hypothyroidism was sustainable for a duration of 72 h with no significant change in LDH activity that significantly increases under euthyroid state (P < 0.001).

Myeloperoxidase activity MPO activity, a marker of neutrophil migration indicates the extent of inflammation. After ischemia/reperfusion euthyroid rats showed significantly elevated MPO activity (P < 0.001) compared to euthyroid controls. Similar degree of MPO activity was observed in hypothyroid ischemic reperfused rats, hypothyroid control and euthyroid control rats when SNK post hoc test was applied (P < 0.001) (Table 2). At 72 h, E + I/R group rats showed a further significant increase of myeloperoxidase activity (P < 0.001), but no further increase was observed in H + I/R group rats indicating that the effect seen was not due to cell death delay. Malondialdehyde levels Significantly higher levels were observed in E + I/R rats as compared to euthyroid controls. SNK post hoc revealed that this difference was primarily due to ischemia reperfusion in euthyroid rats. MDA levels remained unaltered in hypothyroid control and H + I/R rats when SNK post hoc test was performed (P < 0.001) (Table 2). Malondialdehyde levels in E + I/R group showed a further significant increase at 72 h (P < 0.001), but no significant increase was observed in H + I/R group rats indicating that no further peroxidation damage occurred due to delayed cell death.

Nitrite levels Significant difference was observed among all groups. To investigate the difference SNK post Hoc analysis was performed and it was observed that this difference was due to ischemia reperfusion in hypothyroid and euthyroid rats. Levels of nitrite were similar in euthyroid control and hypothyroid control (Table 2). Nitrite levels did not show any significant increase at 72 h in H + I/R group while a significant increase in their levels was observed in E + I/R group (P < 0.001). Mitochondrial alterations Calcium content Significant differences were observed between euthyroid control and euthyroid ischemic rats. To investigate the differences SNK post hoc analysis was performed and it was observed that this difference was primarily due to ischemia/ reperfusion in euthyroid rats. Hypothyroid controls and ischemia reperfused rats were almost similar with respect to calcium parameter. Results are presented in Table 3.

Table 2 Effect of hypothyroidism on biochemical parameters after ischemia following reperfusion at 24 and 72 h a Parameters

No. of animals

Groups E

E + I/R

H

H + I/R

Nitrite (nmol/mg plasma protein)

10

0.46 ± 0.06

0.73 ± 0.13

LDH (U/min/mg plasma protein)

10

0.25 ± 0.07

LDH (U/min/mg tissue protein)

5

4.14 ± 0.44

MPO (U/min/mg tissue protein)

10

0.22 ± 0.06

MDA (nmol/mg tissue protein)

10

0.25 ± 0.05

4.92 ± 1.27 b 8.76 ± 1.11 a, b, c 2.06 ± 0.35 b 2.65 ± 0.49 a, b, c 23.09 ± 4.64 b 33.16 ± 9.21 a, b, c 1.03 ± 0.14 b 2.23 ± 0.52 a, b, c 0.94 ± 0.15 b 1.49 ± 0.13 a, b, c

1.53 ± 0.41 b 1.66 ± 0.63 a, b 0.93 ± 0.25 1.28 ± 0.39 a 9.77 ± 1.90 12.81 ± 3.2 a 0.52 ± 0.01 0.59 ± 0.11 a 0.52 ± 0.08 0.62 ± 0.05

0.33 ± 0.07 7.02 ± 0.39 0.49 ± 0.06 0.53 ± 0.04

Data are presented as mean ± SD. Statistical analysis was performed by one factorial ANOVA and SNK test. E = euthyroid control, H = hypothyroid control, E + I/R = euthyroid ischemic reperfused rat after 24 h, H + I/R = hypothyroid ischemic reperfused rat after 24 h. a Values obtained after ischemia followed by 72 h of reperfusion in their respective groups and number of animals in these groups were 5. b P value <0.05 was considered significant (when ANOVA was applied among groups). c P value <0.05 were considered significant (euthyroid ischemic reperfused group at 24 h vs. 72 h). Power of test is >80%.

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Table 3 Effect of hypothyroidism on biochemical changes and membrane potential in ischemia following 24 h of reperfusion injury Parameters

SOD (U/min/mg protein) Catalase (U/min/mg protein) Membrane potential (% from control) NADPH (AFU) ROS (AFU) Ca2+ i (AFU)

No. of animals

Groups E

E + I/R

H

H + I/R

10 10 10 10 10 10

4.61 ± 1.17 0.53 ± 0.07 100 29.1 ± 1.9 10.08 ± 4.08 1.29 ± 0.11

2.86 ± 0.64* 0.56 ± 0.10 36.3 ± 3.39* 19.6 ± 2.5* 27.2 ± 1.7* 1.9 ± 0.08*

5.14 ± 1.22 0.45 ± 0.06 80.37 ± 6.3 19.9 ± 1.9* 18.72 ± 1.8 1.5 ± 0.14

4.69 ± 0.51 0.51 ± 0.09 62.8 ± 3.9 17.6 ± 2.04* 20.31 ± 1.4 1.59 ± 0.14

AFU denotes arbitrary fluorescence units. Data are presented as Mean ± SD. Statistical analysis was performed by one factorial ANOVA and SNK test. Change in membrane potential is expressed as % from control. *P value < 0.05 were considered significant and Power of test is >80%. E = euthyroid control, H = hypothyroid control, E + I/R = euthyroid ischemic reperfused rat after 24 h, H + I/R = hypothyroid ischemic reperfused rat after 24 h.

Membrane potential The percentage of cells with decreased membrane potential was calculated from the histogram of Rh123 fluorescence intensity. Hypothyroid control and hypothyroid ischemic reperfused rats showed significant retention of membrane potential at a level of 80.37% to 62.8% of euthyroid control. The highly significant decrease in membrane potential in euthyroid ischemic reperfused rats compared to hypothyroid rats in response to reperfusion indicated that hypothyroidism offers a protection against reperfusion injury (E + I/R vs. E P < 0.001, E + I/R vs. H + I/R P < 0.001) (Table 3). Estimation of ROS generation Significant differences were observed among all groups of rats. To investigate the difference SNK post hoc analysis was performed and it was observed that this difference was primarily due to euthyroid ischemic reperfused rats. Hypothyroid control and hypothyroid ischemic rats fell in the same homogenous subset when post hoc test (Student–Newman–Keuls) was applied. Results are presented in Table 3. Energy level (NADPH) Energy levels were significantly depleted in euthyroid ischemic reperfused, hypothyroid control and hypothyroid ischemic reperfused rats in comparison to euthyroid controls (P < 0.001, Table 3). Discussion The present study defines the mechanism of neuronal death through changes in spectrum of biomarkers following the induction of ischemia and reperfusion in a hypothyroid rat brain model. In this study, majority of hypothyroid rats had displayed either the absence of neurological deficit and smaller infarct following ischemia–reperfusion (Fig. 1). Ischemic injuries to brain initiate the cascade of events and change in intracellular metabolites. Therefore, exploration of intracellular mechanisms is important to plan neuroprotective strategies to enhance neuronal survival after cerebral ischemia. These have been explored in initial studies in euthyroid animal models by others (Kristian and Siesjo, 1996; Baker et al., 1998; Kontos, 2001) and individually, these are in agreement with our findings in E + I/R. The elevation of intracellular Ca2+ and decrease in

membrane potential with increased free radical production after MCA occlusion in euthyroid rats was observed. Hypothyroidism leads to a less stressful cellular condition subsequent to ischemia/reperfusion is reflected by non-significant intracellular Ca2+ influx and retention of mitochondrial membrane integrity. This shows that there is less damage to mitochondria in this group (Table 3). In E + I/R group, ROS generation increased by almost 3fold during ischemia reperfusion. Hypothyroidism resulted in only 1.8-fold increase in ROS generation but no further increase was observed due to I/R insult suggesting that further cellular injury was prevented. Though we observed a significant rise in post ischemic/reperfusion in ROS generation under hypothyroid condition, however the extent of increase was significantly less compared to euthyroid condition (Table 3). Significant decrease in SOD activity in euthyroid ischemic group indicated a loss of anti oxidant mechanism necessary for neutralization of reactive oxygen species. The significant decrease in SOD activity further increases the neuronal cell death. SOD activity remains intact under hypothyroid conditions indicating that antioxidant status is better maintained and neutralizes the ROS generated by ischemia/reperfusion. The vector mediated therapeutic modalities for ensuring the sustained superoxide dismutase activity by external means (Keller et al., 1998; Kristian and Siesjo, 1998) seems to be already achieved through hypothyroidism. The many fold significant post ischemic increase in enzymatic activity of LDH, nitrite level, MPO activity and MDA levels indicates severe damage in euthyroid ischemic reperfused rats (Freichs et al., 1990; Rehncrona et al., 1981). Although significant changes were seen in basal MPO activity, MDA levels and ROS under hypothyroid conditions, but cellular integrity was maintained as indicated by a low LDH activity. The concordance of LDH levels estimated in blood collected from the jugular vein of the ipsilateral side, as well as tissue samples confirms secretory origin of these markers from the injury site (Table 2). A 10-fold increase in nitrite content in euthyroid ischemic rats was observed. It was shown that during focal ischemia in rats there was rapid activation of nitric oxide synthase (NOS), which in turn produce injury (Kader et al., 1994). The neuroprotective role of NOS inhibitors in stroke has also been reported (Fassbender et al., 2000). We also observed small

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albeit significant increase in nitrite levels under hypothyroid condition. The nitrite levels were further increased significantly in hypothyroid group after I/R. This increase though substantial, is still many folds lower compared to ischemia–reperfusion injury under euthyroid condition. Though recent studies do implicate increased NOS in offering protection subsequent to primary sub lethal insult (Kiloto et al., 1996; Leker et al., 2001; Zaheer et al., 1997), the NOS threshold leading either to protection or injury is still not known. Though estimations of either inducible NOS (injurious) or endothelial NOS (protective) were not carried out the fact that ROS levels did not increase in H + I/R group suggest the change of nitrite to peroxynitrite radical an unlikely one. This may be the reason for better post ischemic recovery in hypothyroid rats. Normal morphology of neurons was maintained in euthyroid and hypothyroid control rats. Ischemia reperfusion noticeably increases ischemic neurons in E + I/R when compared to hypothyroid ischemic reperfused rats (Fig. 2 panel A). Further we also examined the nature and extent of cell death. The absence of TUNEL-positive cells in euthyroid control, their visibility in hypothyroid control and hypothyroid ischemic reperfused rats in stark contrast to their dominant presence in ischemic–reperfused euthyroid rats is indicative of considerable apoptotic cell death in euthyroid animals (Fig. 2, panel B). These findings are further corroborated by Annexin-V FITC flowcytometry in neuronal preparations from different group of rat brains. The percentage of Annexin-V FITC stained neurons increase significantly subsequent to I/R in euthyroid rats. Under hypothyroid condition, the number of cells dying through apoptosis is significantly higher compared to euthyroid controls. But the dramatic increase observed under euthyroid condition subsequent to I/R is not seen in hypothyroid rats. The significant level of cell death detected by flowcytometery in euthyroid rats seen by us seems to be a combination of both apoptotic (major) and necrotic (minor) components (Fig. 2, panel D). Biochemical, cellular and morphological changes after 24 h suggest that hypothyroidism may offer neuroprotection to rat brain from I/R insult. The issue whether the effect seen by us is a real one and not due to the delayed cell death has been adequately answered by carrying out estimations of various injury related parameters at a longer time point of 72 h. The results indicate that the neuroprotective effect cannot be ascribed delayed cell death (Table 2 and Fig. 1). The severe hypothyroidism may lead to a significant fall in blood pressure, heart rate, and body temperature (Table 1). We did not measure the effect of hypothyroidism on either cerebral blood flow or glucose metabolism in H + I/R rats. Earlier studies have reported that hypothyroidism does not result in any specific local defects in spite of a significant decrease in regional cerebral blood flow (23.4%, P < 0.001) and cerebral glucose metabolism (12.1%, P < 0.001) (Constant et al., 2001). Similarly, a reduction in post-ischemic damage through hypothermia has been explored (Coimbra et al., 1996; Kader et al., 1994). Hypothermia related significant neuroprotection has been observed below 33°C temperature. A few reports do indicate that reduction even by 2°C can cause the decrease in

inflammation in ischemic reperfused animals (Luan et al., 2003). Whether the statistically significant decrease in body temperature of euthyroid and hypothyroid rats (maximum difference being 1.3°C) can explain the many fold neuroprotective changes observed by us in ischemia/reperfused euthyroid and hypothyroid animals is difficult to comprehend. Experiments to assess even the effect of mild hypothermia did not yield any significant changes in either infarct size or biochemical parameters like MDA levels and LDH activity further suggesting that the effect may be purely due to hypothyroidism per se (data not shown). Further studies are warranted to delineate the neuroprotective effects of each these components separately. Whatever may be the contribution of each of these components, the study results clearly show that they are induced by severe hypothyroidism. Our observation that hypothyroidism gives neuroprotection by making brain more tolerant to insult suggests that hypothyroidism affects some intracellular defence mechanism that occurs early in ischemic process. Reduction or slowing down of metabolism has also been shown to confer a remarkable cardio protection in variety of species and causes such as aging, hyperlipidemia and diabetes (Baxter and Ferdinandy, 2001; Ferdinand et al., 1998; Przykleux and Kloner, 1998). Several groups have reported this type of phenomenon leading to curtailment in cell death and injury in ischemic animal models (Ahamed et al., 2000; Kiloto et al., 1996; Kitagawa et al., 1990; Zaheer et al., 1997). Accumulation of neutrophil in focal ischemia has been demonstrated (Barone et al., 1991) and primary insult induced neutrophil infiltration has been implicated to develop tolerance to the further injury (Ahamed et al., 2000). The beneficial effect of hypothyroidism on limiting the size of infarct in animal model is supported by a recent reported study. In a retrospective study on neurological outcome in 761 patients with acute stroke, hypothyroid subjects (serum TSH >10 μU/ml) had significantly better Glasgow Coma Score (P < 0.001) and a trend for better Scandinavian Scale System (P = 0.09). Mean handicap score was significantly better in patient with hypothyroidism at discharge, at 1 month and 1 year (Alevizaki et al., 2005). The present study provides evidence that hypothyroidism makes neuronal tissue less vulnerable to severe ischemic insult. Though the cellular mechanism involved in neuronal protection by hypothyroidism has been investigated, the unequivocal demonstration of primary efficacy of brain-specific hypothyroxinemia mitigating the tissue damage is lacking. The results indicate a need of screening agents that can specifically either reduce the T3 availability to brain or modulate downstream actions of T3. The need for such a study cannot be overstated given the known actions of peripheral hypothyroidism that can lead to confounding effect on interpretation of data. Since severe hypothyroidism may result in marked alteration in body metabolism at various tissue levels and may result in myxedema coma, the strategy of inducing acute hypothyroidism in subjects with stroke cannot be extrapolated in present state of knowledge. However, future studies on identification of agents blocking brain tissue-specific type II monodeiodinase which

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