Intracerebroventricular injection of kisspeptin in male rats activates hypothalamo-pituitary-gonadal axis, but not hypothalamo-pituitary-adrenal axis.

Kisspeptin is an important hormone involved in the stimulation of the hypothalamo-pituitary gonadal (HPG) axis. The HPG axis can be suppressed in certain conditions such as stress, which gives rise to the activation of the hypothalamo-pituitary-adrenal (HPA) axis. However, the physiological role of kisspeptin in the interaction of HPG and HPA axis is not fully understood yet. This study was conducted to investigate the possible effects of central kisspeptin injection on HPG axis as well as HPA axis activity. Adult male Wistar rats were randomly divided into seven groups as followed: sham (control), kisspeptin (50 pmol), P234 (1 nmol), kisspeptin + p234, kisspeptin + antalarmin (0.1 µg), kisspeptin + astressin 2B (1 µg), and kisspeptin + atosiban (300 ng/rat) (n = 10 each group). At the end of the experiments, the hypothalamus, pituitary, and serum samples of the rats were collected. There was no significant difference in corticotropic-releasing hormone immunoreactivity in the paraventricular nucleus of the hypothalamus, serum adrenocorticotropic hormone, and corticosterone levels among all groups. Moreover, no significant difference was detected in pituitary oxytocin level. Serum follicle-stimulating hormone and luteinizing hormone levels of the kisspeptin, kisspeptin + antalarmin, and kisspeptin + astressin 2B groups were significantly higher than the control group. Serum testosterone levels were significantly higher in the kisspeptin kisspeptin + antalarmin, kisspeptin + astressin 2B, and kisspeptin + atosiban groups compared to the control group. Our findings suggest that central kisspeptin injection causes activation in the HPG axis, but not the HPA axis in male rats.

The Impact of the Number of Sutures on Regeneration in Nerve Repair.

PURPOSE: The ideal number of sutures for epineural nerve repair is still unclear. Increased number of sutures increases secondary damage and inflammation to the nerve tissue, which negatively affects nerve regeneration. When the number of sutures decreases, the strength of the nerve repair site decreases and nerve endings are fringed, which also negatively affects nerve regeneration. Therefore, each additional suture is not only beneficial but also detrimental. The aim of this study was to find out the ideal number of sutures for nerve repair. METHODS: Seventy rats were randomly divided into 5 groups. One of the groups was used as a control group, and right sciatic nerves of the rats in other 4 groups were repaired by using 2, 3, 4, or 6 epineural sutures, respectively, after nerve transection. Biomechanical assessment was performed on the nerves collected from these rats at 5 days of follow-up. Functional and histological analyses were evaluated after 12 weeks of follow-up. RESULTS: It was found that an increase in the number of sutures enhances resistance to tensile force in general. However, there was no significant biomechanical difference between the 6-sutured group in which the most sutures were used and the 4-sutured group. In functional examinations, overall successful results were obtained in the group with 4 sutures. In histological examinations, there was no statistical difference between the control group, 2-sutured groups, and 4-sutured groups in terms of connective tissue index. However, it was observed that the group with 6 sutures had a higher connective tissue index than the control group and groups with 2 and 4 sutures. In terms of regeneration index, it was found that repair with 4 sutures was superior to repair with 2 and 6 sutures. No difference was found between any of the suture groups according to the diameter change index. CONCLUSIONS: These results indicate that repair with 4 sutures is the best method of epineural repair that provides both strength and regeneration. These findings will contribute to both the repair of clinically similar nerves and the standardization of rat nerve studies.

Inhibition of Fatty Acid Binding Protein 4 in Obese Male Mice Adversely Affects Reproductive Parameters.

BACKGROUND: As obesity is increasing worldwide, obese people use various methods to get rid of excess weight. BMS309403 (A drug) is a specific inhibitor of fatty acid binding protein 4. In this study, the effects of the BMS309403 on serum biochemical markers, testis tissue spermatogenesis and apoptotic markers were investigated in male mice. METHODS: Balb/c mice (total=56, each group n=14) were divided into control, obese control, obese solvent and obese drug groups. The obese control, obese solvent and obese drug groups were fed on the high sucrose diet to lead to obesity. After the development of obesity, BMS309403 was orally administered to the obese drug group for six weeks. It was performed in testicular tissues (Johnson Score and apoptosis markers) and biochemical tests (total testosterone, sex hormone binding globulin, inhibin-B tests and free androgen index) were used to evaluate reproductive parameters. The p<0.05 was considered to indicate a statistical significance. RESULTS: Serum fatty acid binding protein 4 levels were higher in obese control group and obese solvent group, compared to control (p<0.05) and obese drug groups (p<0.001). Serum total testosterone, free androgen index, inhibin-B, sex hormone binding globulin levels, testicular tissue B-cell lymphoma-2 expression level and Johnson Score parameters were lower in all obese groups compared with the control group. Inhibin-B levels and Johnson Score results were lower in obese drug group compared to other two obese groups (p<0.05). CONCLUSION: Contrary to expectations, the use of BMS309403 negatively affected male reproductive parameters. Negative changes in reproductive parameters may be a result of the increased lee index of obesity.

Neuroprotective Effects of Milrinone on Experimental Acute Spinal Cord Injury: Rat Model.

OBJECTIVE: Spinal cord injury (SCI) disrupts nerve axons with devastating neurological consequences, but there is no effective clinical treatment. The secondary damage mechanism is a mainstay process, and it starts within a few minutes after trauma. We aim to investigate the neuroprotective effects of milrinone on the SCI model. MATERIALS AND METHODS: A total of 36 Wistar albino rats, each weighing 300-400 g, were randomly split into 4 groups that received different treatments: in group 1 (sham) (n = 9) control, only a laminectomy was performed; in group 2 (SCI) (n = 9), SCI was imitated after laminectomy; in group 3 (SCI + saline) (n = 9), physiological saline solution was injected intraperitoneally immediately after the SCI; and in group 4 (SCI + milrinone), milrinone was administered intraperitoneally on lateral decubitus position immediately after the SCI. Spinal cord contusion was established by the weight-drop technique after laminectomy. Neurological examination scores were recorded, and rats were killed 72 hours later. Serum and spinal cord tissue glutathione peroxidase, total antioxidant status, total oxidant status, 8-hydroxiguanosine, interleukin-6 and interleukin-10 levels, histopathological spinal cord damage score, and apoptotic index were examined and compared between groups. RESULTS: Neurological examination scores were significantly better in the milrinone-treated group compared with groups 2 and 3. SCI significantly increased serum and spinal cord tissue glutathione peroxidase, total oxidant status, 8-hydroxiguanosine, and interleukin-6 levels that were successfully reduced with milrinone treatment. Interleukin-10 and total antioxidant status levels decreased as a result of SCI increased with milrinone treatment. Increased histopathological spinal cord damage score and apoptotic index in groups 2 and 3 significantly decreased in group 4. CONCLUSIONS: Milrinone could reduce apoptosis and increase anti-inflammatory and antioxidative mediators, thus playing a protective role in secondary nerve injury after SCI in rats.

Evaluation of Apoptosis Pathway of Geraniol on Ishikawa Cells.

Endometrial cancer is the most common type of cancer in the female reproductive system. Geraniol is acyclic monoterpene alcohol derived from essential oils of aromatic plants. This study aimed to investigate the apoptosis pathway of geraniol on Ishikawa cells. The cytotoxic effects of Geraniol on Ishikawa cells were determined by an MTT test. Ishikawa cells were seeded on cover slips, the IC50 dose was applied, and the cells were incubated with antibodies against Bax, Bcl-2, and TUNEL Assay. mRNA expression analysis of apoptosis-related genes was determined by RT-qPCR with an IC50 dose of Geraniol. The IC50 dose of Geraniol decreased Bcl-2 staining significantly, but it significantly increased Bax staining and TUNEL positive cells. A significant increase in the Bax, caspase3, caspase-8, cytochrome C and Fas genes and a significant decrease in the Bcl-2 gene was observed when the IC50 dose group was compared to the cells in the control group based on their mRNA expression levels.Analysis of expression of genes whose products are involved in apoptosis suggests the involvement of the mitochondrial pathway.

Ameliorating the effects of Adalimumab on rabbits with experimental cerebral vasospasm after subarachnoid hemorrhage.

BACKGROUND: Adalimumab (ADA), which is a new-generation recombinant human monoclonal antibody for tumor necrosis factor α (TNFα), has strong anti-inflammatory effects. The role of enhanced inflammation is well established for the development and progression of cerebral vasospasm. Investigated in the present study is the probable ameliorating and neuroprotective effects of ADA in rabbits using a cerebral vasospasm model with biochemical and histopathological methods. METHODS: Thirty male New-Zealand white rabbits were randomly divided into control, subarachnoid hemorrhage (SAH) only and SAH plus ADA treatment groups. SAH was established as a single cisterna magna autologous arterial blood injection. ADA treatment was started just after intracisternal blood injection and continued for 72 hours once a day. The animals were sacrificed 72 hours after the induction of SAH, serum and brainstem tissue obtained for investigations. RESULTS: Brainstem tissue and plasma levels of tumor necrosis factor-alpha and Interleukin-1ß, brainstem tissue Matrix metalloproteinase-9 levels increased after SAH and partly decreased after treatment. Plasma levels of brain-derived neurotrophic factor decreased after SAH and partly restored after treatment. ADA treatment significantly increased the mean cross-sectional area of the vasospastic basilar arteries, reduced the basilar artery wall thickness and also ameliorates enhanced endothelial apoptosis. CONCLUSION: Findings obtained in this study suggest that ADA is an effective neuroprotective agent for ameliorating cerebral vasospasm in experimental rabbit vasospasm.

Neuroprotective Effects of Tocilizumab on Experimentally-Induced Spinal Cord Ischemia-Reperfusion Injury.

OBJECTIVES: We aimed to evaluate neuroprotective effects of tocilizumab on spinal cord ischemia-reperfusion (I/R) injury. Our study design was an experimental rabbit spinal cord I/R injury model, and the setting was at the Animal Research Laboratory, Necmettin Erbakan University, Meram School of Medicine, Konya, Turkey. METHODS: Twenty-four adult New Zealand rabbits were randomly divided into 3 groups: Group 1, control group (n = 8); Group 2, I/R group, and Group 3 (n = 8) I/R injury + tocilizumab (4 mg/kg, ip) treatment group. Spinal cord I/R injury repair was performed by infrarenal aortic cross clamping. On neurologic evaluation, spinal cord tissue plasma tumor necrosis factor alpha (TNFα), total antioxidant status (TAS), total oxidant status (TOS), thiobarbituric acid reactive substances (TBARS), interleukin 6 (IL-6), interleukin 10 (IL-10) levels were analyzed. Spinal cord neuronal damage score and apoptotic cell count were also investigated. RESULTS: I/R injury significantly increases the plasma and spinal cord tissue TNFα, TOS, TBARS, and IL-6 levels and decreases the plasma and spinal cord tissue TAS and IL-10 levels. Tocilizumab treatment significantly reduces the plasma and spinal cord tissue TNFα, TOS, TBARS, IL-6 levels and increases plasma and tissue TAS and IL-10 levels. I/R injury significantly increases spinal cord neuronal damage score and apoptotic cell count. Tocilizumab treatment significantly reduces spinal cord neuronal damage score and apoptotic cell count. Neurologic examination scores at 24, 48, and 72 hours were significantly better in the treatment group when compared with the I/R group. CONCLUSIONS: This study shows significant neuroprotective effects of tocilizumab on rabbit spinal cord I/R injury.

Investigation of the effect of naringenin on oxidative stress-related alterations in testis of hydrogen peroxide-administered rats.

Testis tissue is prone to oxidation because its plasma membrane contains many polyunsaturated fatty acids. Naringenin is a plant-derived natural flavonoid. We investigated the possible ameliorative role of naringenin on the hydrogen peroxide (H2 O2 )-induced testicular damage in Wistar rats. Animals received 12 mg/kg H2 O2 by intraperitoneal injection, and 50 mg/kg naringenin via orogastric gavage for 4 weeks. In the H2 O2 group, the testis malondialdehyde level increased, while the amount of reduced glutathione, glutathione transferase activities, and the testis weight decreased. There were severe testicular damages in the H2 O2 group otherwise their grade were less in the naringenin + H2 O2 group. However, the serum testosterone concentrations decreased in both the H2 O2 and the naringenin + H2 O2 groups. The testicular zinc and calcium levels reduced in the H2 O2 -treated rats. In conclusion, the administration of H2 O2 caused oxidative stress in the testes and naringenin supplementation decreased the H2 O2 -induced effects, except for changes in testosterone levels.

Telmisartan ameliorates oxidative stress and subarachnoid haemorrhage-induced cerebral vasospasm.

OBJECTIVES: Growing evidence suggests that oxidative stress is one of the factors contributing to subarachnoid haemorrhage (SAH)-induced cerebral vasospasm. SAH-induced cerebral vasospam alters thioredoxin (Trx) cycle enzymes and thioredoxin-interacting protein (TXNIP) as an important endogenous antioxidant system. In this study, we have explored the effects of telmisartan on the vascular morphological changes, endothelial apoptosis, tissue oxidative stress status and the level of Trx cycle enzymes/ TXNIP in a rabbit SAH model. METHODS: Forty male New Zealand rabbits were randomly divided into five groups of eight rabbits each: control group, sham group, SAH group, SAH + vehicle group and SAH + telmisartan group. SAH was created by a single cisterna magna blood injection. SAH + telmisartan group received telmisartan treatment (5 mg/kg intraperitoneal, once daily) for 72 h. The brainstem tissue Trx1, Trx2, Trx reductase (TrxR), TrxR1and TXNIP levels were investigated. Total oxidant status (TOS), total antioxidant status (TAS), malondialdehyde (MDA) levels and tumour necrosis factor alpha (TNF alpha) levels were investigated. Basilar artery segments were investigated for cross-sectional area, wall thickness measurements and endothelial apoptosis. RESULTS: Telmisartan treatment restored the lowered level of Trx1, TrxR, TAS and the expression of TrxR1 seen in SAH. Telmisartan treatment also decreased TXNIP expression, TOS, MDA and TNF alpha levels. Morphological changes of cerebral vasospasm were attenuated after treatment. Endothelial apoptosis significantly reduced. DISCUSSION: Treatment with telmisartan ameliorates oxidative stress and SAH-induced cerebral vasospasm in rabbits. These effects of telmisartan may be associated with downregulation of TXNIP and upregulation of Trx/TrxR.

Effects of Nigella sativa L. seed oil on intima-media thickness and Bax and Caspase 3 expression in diabetic rat aorta.

OBJECTIVE: Hyperglycaemia is an important risk factor for the development and progression of the macrovascular and microvascular complications that occur in diabetes. The expression of apoptotic markers in the aortic medial layer of diabetic rats and the effects of N. sativa L. seed oil on the expression of these markers were investigated in this study. METHODS: Four-month-old adult female Wistar rats (n=21) were divided into 3 groups: Group 1, control; Group 2, diabetes and Group 3, diabetes+N. sativa L. seed oil. Group 3 received 0.2 mg/kg/day N. sativa L. seed (black cumin) oil intraperitoneally 6 days per week for 30 days. At the end of the experiment, abdominal and thoracic aortas of all animals were collected and fixed in 10% formalin solution. Then, 5-µm-thick sections were stained with Verhoeff-Van Gieson stain to evaluate Bax and Caspase 3 expression. Tunica intima-media thickness was measured using the stained sections. RESULTS: There were no significant differences in abdominal or thoracic aortic intima-media thickness among the 3 groups. However, there were significant differences in Bax and Caspase 3 expression in the tunica media of the thoracic and abdominal aortas between Group 1 and Group 2 (p<0.05) and between Group 2 and Group 3 (p<0.05) evaluated with the Kruskal-Wallis and Mann-Whitney U tests. CONCLUSION: It is understood that N. sativa L. seed oil is effective against diabetes. N. sativa L. seed oil is a plant material and has value for further investigation to develop diabetes treatment strategies for preventing apoptosis in vascular structures.

Chemoprotective effect of vitamin E in cyclophosphamide-induced hepatotoxicity in rats.

Cyclophosphamide (CP) has a range of adverse effects on liver tissue in humans and animals. Administering an antioxidant with CP might reduce such side effects. Therefore, we examined the role of vitamin E in CP-induced liver toxicity in rats. Male Wistar albino rats were divided into four groups, each of seven rats: control, CP only, CP + vitamin E, and vitamin E only groups. The rats were administered treatments intraperitoneally for 7 days. Then the serum malondialdehyde (MDA), alanine aminotransferase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) levels were determined while the livers were removed, tissue was prepared using routine histological procedures, sections were stained using hematoxylin and eosin, and the terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) method was applied. Histopathologically, CP caused hydropic degeneration, necrosis, pleomorphism, and mitotic activity. The number of TUNEL-positive cells and the MDA and ALT levels were significantly higher in the CP group. The antioxidant effects of vitamin E significantly decreased the number of TUNEL-positive cells and the ALT and MDA levels, and normalized the liver histopathology. CP induces apoptosis, has toxic effects on liver tissue, and changes the histological structure. The administration of vitamin E prevented the liver tissue damage caused by CP.

Effects of Lipokit® centrifugation on morphology and resident cells of adipose tissue / Efectos de la centrifugación de Lipokit® sobre la morfología y las células residentes del tejido adiposo

The aim of adipose tissue engineering is creating autologus vascularized fat tissue to be used for practical soft tissue reconstruction in human clinic. Unfortunately, in practice, long-term results of fat transplantation are often untrustworthy and unreliable, to overcome this problem different many lipoinjection techniques developed in the last 20 years. Centrifuge is a fundamental stepin the preparation of adipose tissue. We focused on some cell markers especially MSCs markers and histological structural properties after with lipokit centrifugation and without lipokit centrifugation of adipose tissue obtained by liposuction by this new technique. Adipose tissue was taken by liposuction and separates to two portions. One of them is centrifugated by Lipokit machine (C+) has a micro filter and the other is not (C-). After centrifugation smear slides and paraffin sections were prepared from these tissues. These slides were stained with H&E and Toluidine Blue. Paraffin sections were immunohistochemically stained with CD34, von Willebrand Factor, CD73, CD90 and CD105. Smear preparations showed a continuous three dimensional plasma membrane appearance of adipocytes. C+ and C- showed expression of CD34, von Willebrand Factor, CD73, CD90, CD105. C+ seems to have more free cells expressing than C-. While passing the filter of Lipokit, large adipocytes and connective tissue parts disintegrate and thus increases the surface area of lipoaspirate. Lipokit® machine release the group cells which are necessary for angiogenesis and they become more freely to construct angiogenesis.

El objetivo de la ingeniería del tejido adiposo es la creación de tejido graso vascularizado autólogo para ser utilizado en clínica humana para la reconstrucción de tejido blando. Desafortunadamente en la práctica, los resultados a largo plazo del trasplante de grasa son poco fiables y no seguros; para superar este problema, se han desarrollado en diferentes países, en los últimos 20 años, variadas técnicas de lipoinyección. La centrifugación es un paso fundamental en la preparación del tejido adiposo. Nos hemos centrado en algunos marcadores, especialmente, de células precursoras mesenquimales y propiedades histológicas estructurales después de la centrifugación mediante Lipokit y sin la centrifugación por Lipokit del tejido adiposo obtenido mediante liposucción. El tejido adiposo fue tomado por liposucción y se separó en dos porciones. Una se centrifugó mediante el sistema Lipokit (C +), con un microfiltro y la otro no (C-). Después de centrifugación, muestras del frotis y secciones de parafina se prepararon a partir de estos tejidos. Los frotis se tiñeron con H&E y azul de toluidina. Las secciones de parafina se tiñeron inmunohistoquímicamente con CD34, factor de von Willebrand, CD73, CD90 y CD105. Las preparaciones de los frotis mostraron una apariencia tridimensional continua de la membrana plasmática de los adipocitos. Tanto en C+ y C- se observó la expresión de CD34, factor de von Willebrand, CD73, CD90 y CD105. En C+ parecen expresarse más células libres que en C-. Cuando se utilizó el filtro de Lipokit, los adipocitos grandes y partes del tejido conectivo se desintegraron, por lo tanto aumentó el área de superficie de lipoaspirado. El sistema Lipokit® libera los grupos celulares que son necesarios para la angiogénesis y se hacen más libres para promoverla.

Assessment of Spermatozoa Morphology under Light Microscopy with Different Histologic Stains and Comparison of Morphometric Measurements / Evaluación de la Morfología de los Espermatozoides bajo Microscopía Óptica con Diferentes Tinciones Histológicas y Comparación de las Mediciones Morfométricas

The aim was to examine the morphology of spermatozoa with different staining methods and aimed to find the better staining methods for morphology of spermatozoa in our study. Randomized 67 patients taken for the study who were admitted to Assisted Reproductive Techniques Unit. In the first part of the study, smears were stained with Hematoxylin Eosin (HE), Toluidin Blue (TB), Giemsa, Wright, ferrous Weigert haematoxylin stain, Orange G, eosin-aniline blue dye, Shorr Method, Papanicolau, Berg Method, Light Green stain, Acridine Orange (AO) and Janus Green dyes. In the second part of the study, smear preparations of 10 patients with normozoospermic were stained with HE, Toluidin Blue (TB), Shorr Method and Papanicolau. Four measurements were made including the middle piece, head length- head width and tail length for 200 spermatozoa with normal morphology. Comparisons were made between the stains that which showed a better morphology. Condensation assessment was not possible in smears stained with Shorr, Berg, Method, AO. Better assessment of condensation could be made in other stains. In the second part the smallestvalues belonged to of TB stain according to measurements of head of the spermatozoa. There was a significant difference at the head length with TB stain. Although measurements of Shorr and Papanicolau areclose to each otherand the largestvalues belonged to Papanicolau dye. It was concluded that measurement values in human sperm morphology could alter with the used staining method.

El objetivo fue examinar la morfología de los espermatozoides con diferentes métodos de tinción y encontrar los mejores métodos para su estudio. Fueron seleccionados para el estudio, de manera aleatoria 67 pacientes, quienes ingresaron a la Unidad de técnicas de reproducción asistida. En la primera parte del estudio, se realizaron y tiñeron frotis con hematoxilina eosina (HE), Azul de Toluidina (AT), Giemsa, tinción de Wright, Hematoxilina Férrica de Weigert, Anaranjado G, tinción eosina-anilina, método de Shorr, Papanicolau, método Berg, tinción verde brillante, anaranjado de acridina (AO) y tinción verde Janus. En la segunda parte del estudio, se realizaron frotis de 10 pacientes con normozoospérmicos y se tiñeron con HE, AT, Método Shorr y Papanicolau. Se realizaron cuatro mediciones: ancho de la cabeza, longitud de la cabeza, parte media y cola, sobre 200 espermatozoides con morfología normal. Se compararon las tinciones que mostraban mejor la morfología. La evaluación de la compactación no fue posible en los frotis teñidos con los métodos de Shorr, Berg y AO. Una mejor evaluación de la copactación podría hacerse en otras tinciones. En la segunda parte los valores menores correspondieron a la tinción de AT en relación a la medición de la cabeza de los espermatozoides. Hubo una diferencia significativa en la longitud de la cabeza con tinción de AT. Las mediciones en los frotis con técnicas de Shorr y Papanicolau fueron similares, con valores más altos bajo tinción de Papanicolau. Se concluyó que los valores de la medición morfológica en espermatozoides humanos podrían ser alterados según el método de tinción utilizado.

Diabetes mellitus- and cooling-induced bladder contraction: an in vitro study.

The effects of diabetes mellitus during cooling on ACh- and KCl-induced responses were investigated in rat urinary bladder. Diabetes was induced in the rats by 50 mg/kg streptozotocin via an intraperitoneal injection. Rats' body and bladder weights were measured. The isometric tension to ACh (10(-9) - 3 × 10(-4) M) and KCl (5-100 mM) in strips of urinary detrusor muscle of diabetic and non-diabetic rats, in organ baths at 37 and 28ºC were recorded. The body weights were significantly decreased and the bladder weights increased in STZ-induced diabetic group compared to the non-diabetic group. ACh and KCl caused concentration-dependent contractions of urinary bladders from non-diabetic and STZ-induced diabetic rats. During cooling, the sensitivity and the maximal response were significantly higher than those during 37ºC, both in non-diabetic and diabetic preparations. Cooling of detrusor muscle preparations induces a graded contraction inversely proportional to the temperature in diabetic rats. It may be assumed that the cooling response involves the same mechanisms in the diabetic and non-diabetic animals.