Effects of prenatal tobacco smoke on cerebellum histomorphological changes at critical developmental stages in CD-1 mice / Efectos del humo de tabaco en los cambios histomorfológicos del cerebelo en las etapas críticas del desarrollo prenatal en ratones CD-1

SUMMARY: In this study the consequences of prenatal exposure to tobacco smokes on the histo-morphological changes of cerebellum was assessed by comparing the smoker mice to the nonsmoker mice. A total of 30 pregnant cd-1 mice were divided into three groups of 10 mice each and with two replicates per group (5 mice each). Following acclimation for five days, the mice were placed in a special modified smoking machine for 2 hours per day over a two- and three-week period for group two and group three, respectively. Group one was considered as a control group. Mice in the control group were exposed simultaneously to fresh air from the room, while those in the treatment groups were exposed to tobacco smoke from six commercial filter cigarettes, containing 0.8 mg of nicotine, 10 mg of tar, and 10 mg of carbon monoxide, for three 1-hour exposure periods every day for three weeks. The mice in the control group were exposed to room air for three 1-hour periods every day for the same period of three weeks. The results from this study showed a correlation between maternal smoking and histological changes in Neuron purkinjense (Purkinje cells) of the cerebellum. They also showed that prenatal smoking period may have caused more damage in the histology and structure of Neuron purkinjense in some juvenile mice. An increased incidence of morphology damage of the cerebellum's Neuron purkinjense' structures was also observed in fetuses with prolonged exposure to tobacco smoking. Exposure of in utero maternal smoking may interfere with brain biological development parameters, giving rise to structural abnormalities of the cerebellum. This study concluded that tobacco smoke exposure to pregnant mice may affect neurodevelopment which may induce behavioural changes as a result of reduced cerebellar size and function.

RESUMEN: Se evaluaron los efectos producidos por la exposición prenatal al humo de tabaco en ratones expuestos y no expuestos y los cambios histomorfológicos observados en el cerebelo en ambos grupos. Un total de 30 ratones cd-1 preñados se dividieron en tres grupos de 10 ratones cada uno y con dos réplicas por grupo (5 ratones cada uno). Después de la aclimatación durante cinco días, los ratones se colocaron en una máquina de fumar modificada, especial durante 2 horas al día, durante un período de dos y tres semanas para el grupo dos y el grupo tres, respectivamente. El grupo uno se consideró como grupo control. Los ratones del grupo de control fueron expuestos simultáneamente al aire limpio de la habitación, mientras que los grupos de tratamiento fueron expuestos al humo de tabaco de seis cigarrillos comerciales, que contenían 0,8 mg de nicotina, 10 mg de alquitrán y 10 mg de monóxido de carbono. durante tres períodos de 1 hora diariamente, durante tres semanas. Los ratones del grupo de control se expusieron al aire ambiente durante tres períodos de 1 hora todos los días durante el mismo período de tres semanas. Los resultados de este estudio mostraron una correlación entre el tabaquismo materno y los cambios histológicos en las neuronas purkinjenses (células de Purkinje). Se observó además que el período de tabaquismo prenatal puede haber causado mayor daño en la histología y estructura de las neuronas purkinjenses en algunos ratones jóvenes. También se observó una mayor incidencia de daño morfológico de las estructuras de las neuronas purkinjenses del cerebelo en fetos con exposición prolongada al tabaquismo. La exposición al tabaquismo materno en el útero puede interferir con los parámetros de desarrollo biológico del cerebro, dando lugar a anomalías estructurales del cerebelo. Este estudio concluyó que la exposición al humo del tabaco en ratones preñados puede afectar el desarrollo neurológico, lo que puede inducir cambios de comportamiento como resultado de la reducción del tamaño y la función del cerebelo.

Immunoexpression of vascular endothelial growth factor, beta-cell lymphoma 2 and cluster of differentiation 68 in cerebellar tissue of rats treated with Ganoderma lucidum / Inmunoexpresión del factor de crecimiento endotelial vascular, linfoma de células beta 2 y grupo de diferenciación 68 en tejido cerebeloso de ratas tratadas con Ganoderma lucidum

Traumatic brain injury (TBI) can potentially lead to hemorrhages in all areas of the skull, which can damage cells and nerve connections. This study aims to investigate the protective effects of Ganoderma lucidum polysaccharides (GLPS) as a antioxidant on cerebellar cell tissues after traumatic brain injury in rats. Sprague Dawley rats were subjected to TBI with a weight-drop device using 300 g1m weight-height impact. The groups are consisted of control, trauma, and trauma+Ganoderma lucidum groups. At seven days post-brain injury, experimental rats were decapitated after intraperitoneal administration of ketamine HCL (0.15 ml/100 g body weight). Cereballar samples were taken for histological examination or determination of malondialdehyde (MDA) and glutathione (GSH) levels and myeloperoxidase (MPO) activity. Significant improvement was observed in cells and vascular structures of Ganoderma lucidum treated groups when compared to untreated groups. It is believed that Ganoderma lucidum may have an effect on the progression of traumatic brain injury. Ganoderma lucidum application may affect angiogenetic development in blood vessel endothelial cells, decrease inflammatory cell accumulation by affecting cytokine mechanism and may create apoptotic nerve cells and neuroprotective mechanism in glial cells.

La lesión cerebral traumática (LCT) puede provocar hemorragias en todas las áreas del cráneo, lo que puede dañar las células y las conexiones nerviosas. Este estudio tuvo como objetivo investigar los efectos protectores de los polisacáridos de Ganoderma lucidum (GLPS) como antioxidante en los tejidos de las células del cerebelo después de la lesión cerebral traumática en ratas. Ratas Sprague Dawley fueron sometidas a TBI con un dispositivo de caída de peso usando un impacto de peso de 300 g-1 m. Se formaron los siguientes grupos: control, trauma y trauma + Ganoderma lucidum. Siete días después de la lesión cerebral, las ratas experimentales fueron decapitadas después de la administración intraperitoneal de ketamina HCL (0,15 ml / 100 g de peso corporal). Se tomaron muestras cerebrales para el examen histológico y para la determinación de niveles de malondialdehído (MDA) y glutatión (GSH) y actividad de mieloperoxidasa (MPO). Se observó una mejora significativa en las células y las estructuras vasculares de los grupos tratados con Ganoderma lucidum en comparación con los grupos no tratados. Durante el estudio se observó que Ganoderma lucidum puede tener un efecto sobre la progresión de la lesión cerebral traumática. La aplicación de Ganoderma lucidum puede afectar el desarrollo angiogénico en las células endoteliales de los vasos sanguíneos, disminuir la acumulación de células inflamatorias al afectar el mecanismo de las citocinas y puede crear células nerviosas apoptóticas y un mecanismo neuroprotector en las células gliales.

Neuroprotective effect ofBacopa monnieri against morphine-induced histopathological changes in the cerebellum of rats

Opioid addiction is associated with oxidative cell injury in neuronal cells. In this study, Bacopa monnieri [L.], a reputed nootropic plant, was evaluated against morphine-induced histopathological changes in the cerebellum of rats. B. monnieri methanolic extract [mBME] [40 mg/kg, p.o] and ascorbic acid [50 mg/kg, i.p] were administered two hours before morphine [20 mg/kg, i.p] for 14 and 21 days. The in vitro antioxidant activity of mBME was determined by 2,2-diphenyl-1-picrylhydrazyl [DPPH] free-radical scavenging assay. Morphine produced vacuolization of basket and stellate cells and reduced the size of Purkinje cells in the cerebellum after 14 days. However, treatment for 21 days was associated with severe shrinkage of Purkinje cells with loss of their characteristic flask-shaped appearance as well as degeneration of basket, stellate and granule cells. Pretreatment with mBME and ascorbic acid for 14 and 21 days attenuated the morphine-induced histopathological changes in the cerebellum. The EC50 for the DPPH free-radical scavenging assay of mBME [39.06 [microg/mL] as compared to ascorbic acid [30.25 microg/mL] and BHT [34.34 microg/mL] revealed that mBME strongly scavenged the free-radicals and thus possessed an efficient antioxidant propensity. These results concluded that B. monnieri having strong antioxidant activity exerted a protective effect against morphine-induced cerebellar toxicity

Morphological and immunohistochemical analysis of apoptosis in the cerebellum of rats subjected to focal cerebral ischemia with or without alcoholism model

ABSTRACT PURPOSE: To evaluated histopathological changes, morphometric and expression of proteins CASPASE-3, BCL-2 and XIAP related to apoptosis in the cerebellum after induction of temporary focal cerebral ischemia followed by reperfusion, with or without a model of chronic alcoholism. METHODS: Fifty Wistar rats were used and divided into: control group (C), sham group (S), ischemic group (I), alcoholic group (A), and ischemic and alcoholic group (IA). The cerebellum samples collected were stained for histopathological and morphometric analysis and immunohistochemistry study. RESULTS: Histopathological changes were observed a greater degree in animals in groups A and IA. The morphometric study showed no difference in the amount of cells in the granular layer of the cerebellum between the groups. The expression of CASPASE-3 was higher than BCL-2 and XIAP in the groups A and IA. CONCLUSION: We observed correlation between histopathological changes and the occurrence of apoptosis in cerebellar cortex.

[Effect of Monosodium glutamate on rat cerebellum]

Background and Objective: Monosodium glutamate [MSG] is used as a food additive. Several studies have reported the adverse effects of Monosodium glutamate on the testis and brain. This study was performed to determine the effect of Monosodium glutamate in rat cerebellum

Methods: In this experimental study, 24 adult wistar rats randomly allocated into three groups including experiment A, experiment B and control [C]. The animals in experiment A and B were received 3g and 6g of MSG thoroughly mixed with their feeds for 14 days, respectively. Animals in control group were received MSG free diet. Food and water for rats to be free in all of experimental time. The rats were sacrificed on fifteen day. The cerebellum dissected and fixed with formalin 10% buffer and stained with hematoxylin and eosin

Results: Disorders and detachment were observed in Purkinje and granular cell layers. Neural cell distribution in granular layer redeuced in the experimental groups. Cellular degenerative changes in the granular layer of the experimental B were more severe than experimental group A. The mean number of neuron of the granular layer in the experimental A, B and control groups were 2750, 2140 and 3150, respectively

Conclusion: The consumption of monosodium glutamate dose dependly causes histopathological changes and reduces the number of the cerebellumllar neurons in adult rat

Depletion of body and cerebellar weights after chronic lithium carbonate ingestion in Albino rats

The effect of Lithium on the body and brain are well known but enough literature is not available on the effects of Lithium on body and Organ weights. So this study was undertaken to see the effect of Lithium on body and cerebellar weights. An experimental study. This study was conducted at the Anatomy Department BMSI, JPMC, Karachi. April 2012 to June 2012. Thirty male albino rats of 200-250 grams were selected and divided into two major groups A and B. Each major group consisted of 15 animals each groups was further divided into three sub-groups according to the time period of the study which was 2 weeks, 6 weeks and 12 weeks. Group A served as control group which was given lab diet and B was the Lithium treated group. Lithium carbonate was given at a dose of 20mg/kg/day for 2, 6 and 12 weeks. Body weights were recorded at the start and of each time period, but cerebellar, Organ weights were recorded at the end of 2[nd], 6[th] and 12[th] weeks. The body weights and Organ weights are recorded on digital electronic weighing scale. Group B showed a progressive decrease of body and cerebellar weights as the time period of study advanced. But relative cerebellar weights of group B increased with increasing time period of study. The present study concluded that Lithium carbonate causes a significant decrease of body and cerebellar weights

[Excitatory and inhibitory effects of nitric oxide on weight, size, and histological changes of rat cerebellum]

Nitric oxide [NO] is produced in different body organs in mammals and numerous physiological and pathological properties are attributed to this small molecule. The precursor of this substance in the body, L-arginine, is synthesized by the enzyme nitric oxide synthase [NOS], and it is catalyzed, and is inhibited by a substance called L-NG-nitroarginine methyl ester [L-NAME]. In this study we investigated the qualitative and quantitative effects of nitric oxide on cerebellar histopathology in vivo environment via increasing and decreasing its production. Forty Wister rats, weighing 200- 250 gr with a mean age of 8 weeks, were divided into 5 groups after making sure the rats were pregnant. Except the control group, the other pregnant groups, respectively received: 2 ml/kg normal saline, 200 mg/kg L-arginine, 20 mg/kg L-NAME and a mixture of the same doses of L-arginine and L-NAME on the third, fourth and fifth days of pregnancy. On day 18 of pregnancy, we anesthetized the rats, excised the cerebellum after craniotomy and fixed the organs in 10% formalin. We later prepared 5 to 6-micron in thickness tissue sections and dyed them by the routine Hematoxylin and eosin [HE] and Masson's Trichrom staining methods before studying them by light microscopy. There was a significant difference between the rats receiving L-arginine and the rats in other groups [P<0.01]. This study showed that L-NAME is capable of significantly decreasing the injury caused by nitric oxides in rat cerebellum

Phenytoin-induced Toxicity in the Postnatal Developing Cerebellum of Wistar Rats, Effect of Calotropis procera on Histomorphometric Parameters / Toxicidad Inducida por Fenitoína en el Desarrollo Postnatal del Cerebelo de Ratas Wistar, Efecto de Calotropis procera sobre los Parámetros Histomorfométricos

The role of methanolic leaf extracts of Calotropis procera in phenytoin-induced toxicity on histomorphometric variables in the postnatal developing cerebellum of Wistar rat was studied. Pregnant rats were treated orally with 50 mg/kg phenytoin in pre and post natal life and 300 mg/kg methanolic leaf extract of Calotropis procera 1 hour prior to phenytoin administration. 200 mg/kg vitamin C (standard antioxidant) was also administered orally 1 hour prior to phenytoin treatment. The control animals received water. Standard diet of rat pellets and water were provided ad libitum. At the end of the experiment, the offspring of days 1, 7, 14, 21, 28 and 50 post partum, five per group were sacrificed by cervical dislocation. The cerebellum of all groups were dissected out and processed for histomorphometric studies. The results showed in the developing cerebellum of phenytoin treated animals, a delayed cell maturation in the external granular layer, reduction of the molecular layer, astrocytic gliosis and loss of Purkinje cells on day 50 postpartum. Administration of extracts of Calotropis procera and vitamin C though reversed these changes when compared with the phenytoin treated group, but not significantly when compared with the control. In conclusion, supplementation with methanolic extracts of Calotropis procera reduced the rate at which phenytoin induced toxicity in the postnatal developing cerebellum of Wistar rat.

Fue estudiado el rol de los extractos metanólicos de las hojas de Calotropis procera en la toxicidad inducida por fenitoína sobre las variables histomorfométricas en el desarrollo postnatal del cerebelo de ratas Wistar. Ratas preñadas fueron tratadas por vía oral con 50 mg/kg de fenitoína durante la vida pre y post natal. Además, fue administrado, por vía oral, una hora antes del tratamiento con fenitoína 300 mg/kg de extracto metanólico de las hojas de Calotropis procera y 200 mg/kg de vitamina C (antioxidante estándar). Los animales control recibieron agua. Una dieta estándar de pellets para rata y agua se proporcionaron ad libitum. Al final del experimento, 5 crías por grupo de 1, 7, 14, 21, 28 y 50 días post parto, fueron sacrificadas por dislocación cervical. El cerebelo de todos los animales de los diferentes grupos fueron disecados y procesados para el estudio histomorfométrico. Los resultados mostraron en el desarrollo del cerebelo de los animales tratados con fenitoína un retraso en la maduración de células en la capa granular externa, reducción de la capa molecular, gliosis astrocitaria y pérdida de las células de Purkinje en el día 50 post parto. La administración de extractos de Calotropis procera y vitamina C, aunque invirtieron estos cambios, en comparación con los grupos tratados con fenitoína, no fueron significativos en comparación con el control. En conclusión, la suplementación con extractos metanólicos de Calotropis procera redujo la velocidad a la que la fenitoína induce toxicidad en el desarrollo postnatal del cerebelo de ratas Wistar.

Histomorphologic Alterations of the Cerebellum of Wistar Rats following Amodiaquine Plus Artesunate Administration

Amodiaquine and artesunate are two antimalarial drugs sold in combination as Larimalr'. This drug is a very effective artemisinin-base combination. This study was to access the effects of amodiaquine and artesunate combination on the histology of the cerebellum. Twenty adult Wistar rats weighing between 150-180g were divided into four groups (A; B; C and D) of five animals each. Group A served as the control and the animals received distilled water; while group B received 8.75+2.86mg/kg of amodiaquine and artesunate combination for three days; group C received 8.75+2.86mg/kg of amodiaquine and artesunate combination for six days and group D received 17.50+5.71mg/kg of amodiaquine and artesunate combination for three days. Histological sections showed destruction of the Purkinje cortical layers in group B; with increased destructions in groups C and D compared to the control. These results reveal that amodiaquine and artesunate combination causes histological alterations; which were dose and time dependent and these may result in cerebellar dysfunction

Neuroprotective effects of zinc on antioxidant defense system in lithium treated rat brain.

With a view to find out whether zinc affords protection against lithium toxicity the activities of antioxidant enzymes and lipid peroxidation profile were determined in the cerebrum and cerebellum of lithium treated female Sprague Dawley rats. Lipid peroxidation was significantly increased in both the cerebrum and the cerebellum of animals administered with lithium for a total duration of 4 months as compared to the normal control group. On the contrary, the activities of catalase and glutathione-s-transferase (GST) were significantly reduced after 4 months of lithium treatment. The activity of superoxide dismutase (SOD) was significantly increased in the cerebrum after 4 months lithium administration, whereas in the cerebellum the enzyme activity was unaffected. No significant change in the levels of reduced glutathione (GSH) was found in either cerebrum or cerebellum after 2 months of lithium treatment. However, 4 months lithium treatment did produce significant changes in GSH levels in the cerebrum and in the cerebellum. Zinc supplementation for 4 months in lithium-treated rats significantly increased the activities of catalase and GST in the cerebellum, showing that the treatment with zinc reversed the lithium induced depression in these enzyme activities. Though, zinc treatment tended to normalize the SOD activity in the cerebrum yet it was still significantly higher in comparison to normal levels. From the present study, it can be concluded that the antiperoxidative property of zinc is effective in reversing the oxidative stress induced by lithium toxicity in the rat brain.

Arsenic induced free radical toxicity in brain of mice.

The present study was designed to investigate the in vivo effects of oral administration of arsenic trioxide (As2O3; 0.5 and 1 mg/kg body weight/day for 45 days) on cerebral hemispheres and cerebellum in male mice, Mus musculus. Arsenic reduced the concentration of glutathione (GSH) in cerebral hemisphere and cerebellum at both the dose levels; while increased lipid peroxidation (LPO) in cerebral hemisphere and cerebellum regions. Further, the activities of antioxidant enzymes viz., superoxide dismutase and catalase also declined in these two regions with dose indicating oxidative stress. This effect is caused by the action of reactive oxygen species (ROS) induced by arsenic exposure.

Neurotoxicological effects of steroidal compounds in changes of cholesterol level on albino rat brain

Changes in the level of cholesterol in albino rat brain [cerebrum hemisphere, cerebellum and brain stem] were evaluated following the administration of four steroidal derivatives as 3 beta-Acetoxy-5-bromo 6-beta succinimido, 5 alpha-cholestane A[I], 3 beta-chloro-5-bromo 6 beta-amino-5 alpha-cholestane B[II], 3 beta-Acetoxy-6-phthalimid choloest-5-ene C[III] and 6-nitro cholest 5-eno [3 alpha-4 alpha-d] oxazolidin-2-one D[IV]. Experiments were carried out on four groups of six male albino rats weighing 200 +/- 20gm. Control rats were administered normal saline water intraperitoneally [ip] while the other experimental groups were given 0.3 mg/kg bodyweight of steroidal solution which was prepared in peanut oil

Effect of cerebellar modulation on rat gastric secretion and enterochromaffin-like cell.

Effect of cerebellar lesion and vestibular stimulation (VS) on the activity and alternation of ECL-cells along with changes in gastric volume and acid secretion was studied. The results suggest that cerebellar lesion caused increased gastric volume and acid secretion and tended to decrease ECL-cell density. On the other hand VS of nodular lesioned rats resulted in decrease of above parameter which became marked only after 21 days of nodular lesion.

Changes of gastroduodenal peroxidase activity of rats by cerebellar modulation.

The effect of vestibulo-cerebellar lesion and its stimulation by rotation on gastric and duodenal peroxidase activity of rats was studied. Vestibulocerebellar lesion by kainic acid produced gastroduodenal ulceration and peroxidase activity of these tissues were decreased. Mucosal thickness of gastric and duodenal tissue were also decreased. It was observed that when vestibulo-cerebellar lesioned rats were subjected to vestibular stimulation, the peroxidase activity was increased together with increased mucosal thickness of gastric and duodenal tissue. At the same time, it was noted that the severity of ulceration was decreased. We conclude that the study of peroxidase activity is a sensitive and potentially useful estimate of gastric and duodenal injury produced by cerebellar lesion that can be valuable in assessing ulcerogenesis and healing.

Diethylhydroxylamine given in vivo inhibits lipid peroxidation and lipofuscin formation in the nervous tissues of rat.

In vivo effects of diethylhydroxylamine (DEHA) on lipid peroxidation and lipofuscin formation in the nervous tissues of rat have been investigated. Rats were fed DEHA for 30, 60 and 90 days and lipid peroxidation levels and lipofuscin concentration measured in cerebellum, brain stem and spinal cord. Lipofuscin contents were also assessed histochemically. The results showed that the drug caused a significant reduction in lipid peroxidation level and lipofuscin concentration related to ageing.

Effect of chronic phenobarbitone toxicity on the cerebellum of adult rats: an electron microscopic study

The degenerative effect of chronic phenobarbitone toxicity on the structure of the adult rat cerebellum was studied in this investigation. 24 adult male albino rats were used, 16 of them were daily injected i.m. with 0.02 mg/kg body weight of phenobarbitone for 3 weeks. The remaining rats were injected with sterilized water [control group]. The body weights were recorded before the first injection and after the last one. The animals were killed by intracardiac perfusion of 20 ml of isotonic saline followed by 250 ml of 2.5% gluteraldehyde. The brain of these animals were dissected. The total brain and cerebellar weights were determined. The ratio of cerebellar to brain weight was calculated for each animal. The pyramis vermis of the cerebellum was dissected out and processed for transmission electron microscopy [TEM]. The results revealed that there was a highly significant retardation of body, brain and cerebellar growth of treated animals compared with the control group. There was also a highly significant decrease in the ratio between cerebellar and brain weights of those animals compared with the control group. TEM examination revealed evidence of degeneration in the mitochondria and myelin. Mitochondrial degeneration varied from swelling, fragmentation and disorganization of cristae to complete granulation and, finally, total breakdown of mitochondrial contents. The nuclei are irregular and contain dense chromatin mostly peripheral in position. Myelin degeneration was expressed in scattered regions, where the axonal myelin sheath was made up of dense staining lamellae. Irregularities in membrane wrapping, segmented swelling and disruption of lamellae, uneven staining and spaces within the myelin sheath were manifested such degenerations were found mostly in the granule cell layer

Methyl parathion induced regional alterations in the regulatory proteins during critical stage of central nervous system development in albino rat pups.

Sublethal doses of methyl parathion (O,O-dimethyl-O-nitrophenyl- thiophosphate) injected intraperitoneally to 15 and 21 day old rat pups induced regional alterations in the central nervous system (CNS) in the levels of total RNA, total proteins, modulatory protein Calmodulin (CaM), in the activity levels of membrane bound enzyme Ca(2+)-ATPase and phospholipids. Levels of RNA and total proteins increased considerably in 15 days old methyl parathion treated (MPT) rat pups. Contrary to this the RNA and total protein content exhibited remarkable decrease in 21 day old methyl parathion treated animals. Calmodulin level showed an increase in cerebral cortex and brain stem and decrease in cerebellum and spinal cord in 15 day old methyl parathion treated rat pups. Whereas the level of Calmodulin decreased in cerebral cortex and cerebellum and increased in brain stem and spinal cord in 21 day old methyl parathion treated rat pups. Activity levels of calcium dependent ATPase showed significant inhibition in all the regions of Central Nervous System (CNS) of 15 and 21 day old methyl parathion treated rat pups. Phospholipids showed a general increase in all the regions of Central Nervous System on methyl parathion exposure. In the light of these observations, it has been suggested that the molecular regulatory mechanisms involving Ca2+/CaM are rendered inefficient due to toxic impact of methyl parathion.

Dermal toxicity of hexachlorocyclohexane (HCH) in rabbit.

Application of HCH (25 mg/kg) on dorsal, ventral and thigh regions of the skin of male rabbits resulted in poisoning and mortality of animals. Morphological changes in skin, liver, kidney, testes and cerebellum together with highly significant alterations in serum and liver enzymatic activity and residue in blood suggested that absorption of HCH and its toxicity could be severe when the pesticide comes in contact with the skin of thigh region of body.