Amniotic membrane reduces wound size in early stages of the healing process.

OBJECTIVE: To investigate the effects of dispase de-epithelialised, glycerol cryopreserved amniotic membrane (AM) on full-thickness skin defects, using a rat model. METHOD: Skin defects of 15 mm diameter were surgically created and measured on the scalps of 53 male rats. Animals were divided into two groups and followed for 0, 3, 7, 14 or 21 days. AM group wounds were covered with de-epithelialised AM and sodium chloride-moistened Aquacel (ConvaTec Inc.); control group wounds were covered with sodium chloride-moistened Aquacel alone. After the follow-up, wounds were measured again, serum samples were taken and wound sites were harvested for histological analysis. Systemic interleukin-4 (IL-4) levels were analysed from serum. RESULTS: On day 3, a statistically significant difference (p < 0.01) was observed in mean wound size, with wound size in the AM group smaller than in the control group (60 ± 12% vs 81 ± 13% of the original size); other time points showed no significance difference in wound size between the two groups. We could not detect differences between the groups in histological parameters or serum IL -4 levels. CONCLUSION: According to this study, AM enhances early stage wound healing in terms of wound size but its effect decreases in later phases. The IL-4 results provide no clear evidence that IL-4 contributes to the effect of AM on wound healing. DECLARATION OF INTEREST: This study was financially supported by the Competitive Research Funding of the Tampere University Hospital (Grant 9H041, 9J047). The authors have no additional conflicts of interest to declare.

Threefold exposure to moderate hypobaric hypoxia decreases the expression of Cu,Zn-superoxide dismutase in some regions of rat hippocampus.

The effect of moderate hypobaric hypoxia on the expression of a peptide antioxidant Cu,Zn-superoxide dismutase in rat hippocampal neurons was evaluated in an immunocytochemical study. The expression of Cu,Zn-superoxide dismutase decreased significantly in the dorsal hippocampus (CA1 and CA2) and tended to decrease in ventral regions (CA3 and dentate gyrus) by the 24th hour after 3-fold exposure to hypoxia.

[Changes in Mn-superoxide dismutase expression in rat hippocampus induced by single and triple exposure to moderate hypobaric hypoxia].

The purpose of this work was to study the dynamics of expression of mitochondrial Mn-dependent superoxide dismutase (Mn-SOD) 3 and 24 hours after single and triple exposure to mild hypoxia. The investigation was conducted in 18 male Wistar rats using immunocytochemical method. It was shown that in various hippocampal areas the effects of single and triple hypoxia exposure on the Mn-SOD expression could be different or largely similar. The expression dynamics at four time points studied (3 and 24 hours after the first exposure, 3 and 24 hours after the third one) had a wave character which may be important for the development of moderate hypoxia-induced tolerance to subsequent more severe exposures.

Expression of beta-nerve growth factor and its receptor in rat seminiferous epithelium: specific function at the onset of meiosis.

beta-Nerve growth factor (NGF) is expressed in spermatogenic cells and has testosterone-downregulated low-affinity receptors on Sertoli cells suggesting a paracrine role in the regulation of spermatogenesis. An analysis of the stage-specific expression of NGF and its low affinity receptor during the cycle of the seminiferous epithelium in the rat revealed NGF mRNA and protein at all stages of the cycle. Tyrosine kinase receptor (trk) mRNA encoding an essential component of the high-affinity NGF receptor was also present at all stages. In contrast, expression of low affinity NGF receptor mRNA was only found in stages VIIcd and VIII of the cycle, the sites of onset of meiosis. The low-affinity NGF receptor protein was present in the plasma membrane of the apical Sertoli cell processes as well as in the basal plasma membrane of these cells at stages VIIcd to XI. NGF was shown to stimulate in vitro DNA synthesis of seminiferous tubule segments with preleptotene spermatocytes at the onset of meiosis while other segments remained nonresponsive. We conclude that NGF is a meiotic growth factor that acts through Sertoli cells.

Thioredoxin-1 expression levels in rat hippocampal neurons in moderate hypobaric hypoxia.

Previous studies have demonstrated that preconditioning (PC) with three sessions of moderate hypoxia significantly increases the expression of the antioxidant protein thioredoxin-1 (Trx-1) in the rat hippocampus by 3 h after subsequent acute severe hypoxia as compared with non-preconditioned animals. However, it remained unclear whether this increase in Trx-1 accumulation during PC is induced before severe hypoxia or is a modification of the response to severe hypoxia. This question was addressed in the present investigation using experiments on 12 adult male Wistar rats with studies of Trx-1 expression after PC without subsequent severe hypoxia. Immunocytochemical studies were performed 3 and 24 h after three episodes of moderate hypobaric hypoxia (three sessions of 2 h at 360 mmHg with 24-h intervals). Immunoreactivity to Trx-1 24 h after the last session was significantly decreased in neurons in all the areas of the hippocampus studied (CA1, CA2, CA3, and the dentate gyrus). Immunoreactivity in CA3 was also decreased 3 h after hypoxia. These results provide evidence that moderate preconditioning hypoxia itself not only does not increase, but even significantly decreases Trx-1 expression. Thus, increases in Trx-1 contents in the hippocampus of preconditioned animals after severe hypoxia are not associated with the accumulation of this protein during PC, but with a PC-induced modification of the reaction to severe hypoxia.

Prominent expression of acidic fibroblast growth factor in motor and sensory neurons.

Several growth factors originally characterized and named for their action on a variety of cells have more recently been suggested to be importantly involved in the development and maintenance of the nervous system. Acidic fibroblast growth factor (aFGF) is a member of a family of seven structurally related polypeptide growth factors. The cells responsible for expression of aFGF in the nervous system of adult rats have been identified using an affinity-purified antibody to aFGF in immunohistochemical studies and synthetic oligonucleotide probes for in situ hybridization studies. High levels of aFGF expression were observed in motoneurons, primary sensory neurons, and retinal ganglion neurons. Glial cells did not express detectable amounts of aFGF. Confocal and electron microscopic analysis suggested that a large portion of aFGF immunoreactivity was associated with the cytoplasmic face of neuronal membranes, consistent with the hypothesis that aFGF is a sequestered growth factor.

Taurine reduces caspase-8 and caspase-9 expression induced by ischemia in the mouse hypothalamic nuclei.

Taurine is a sulphur-containing amino acid abundant in the nervous system. It protects cells from ischemia-induced apoptosis, but the mechanism underlying this is not well established. The aim of our study was to explore the effects of taurine on two main pathways of apoptosis induced by ischemia: receptor-mediated and mitochondrial cell death. Brain slices containing the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus were incubated in vitro under control and simulated ischemic (oxygen-glucose deprivation for 30 min) conditions in the absence and presence of 20 mM taurine. Brain slices were harvested after the 180-min "postischemic" period and fixed in 4% paraformaldehyde. To estimate apoptosis, immunostaining was done for caspase-8 and caspase-9 in paraffin-embedded sections. Immunoreactive caspase-8 and caspase-9 cells were observed in SON and PVN in all experimental groups, but in the "ischemic" group the expression of caspase-8 and caspase-9 and the number of immunoreactive cells was significantly increased in both hypothalamic nuclei. Addition of taurine (20 mM) to the incubation medium induced a marked decrease in caspase-8 and caspase-9 immunoreactivity after ischemia in SON and PVN when compared with the taurine-untreated "ischemic" group. Taurine reduces ischemia-induced caspase-8 and caspase-9 expression, the key inductors of apoptosis in SON and PVN.

[Thioredoxin-1 expression level in hippocampal neurons of rats exposed to hypobaric hypoxia].

It was shown recently that preconditioning by 3-time repetitive mild hypoxia significantly augmented expression of thioredoxin-1 (Trx-1) antioxidant protein at 3 h after subsequent acute severe hypoxia in rat hippocampus as compared to the expression of this protein in non-preconditioned rats. However, it was unclear whether this augmentation was due to an accumulation of Trx-1 during the preconditioning before severe hypoxia or by a modification of reaction to severe hypoxia itself. To answer this question, Trx-1 expression level after preconditioning without subsequent severe hypoxia was studied in an experiment on 12 mature male Wistar rats. Trx-1 expression was studied by immunocytochemistry 3 h and 24 h after thrice-repeated mild hypobaric hypoxia (three 2h treatments at 360 Torr spaced by 24 h intervals). 24 h after the last hypoxic treatment, Trx-1 immunoreactivity was significantly decreased in the neurons of all the hippocampal regions studied (CA1, CA2, CA3 and dentate gyrus). In CA3 it was also decreased at 3 h after hypoxia. These findings indicate that mild preconditioning hypoxia, by itself, does not increase, but on the contrary, decreases Trx-1 expression. It may be concluded that the augmentation of Trx-1 content in the preconditioned animals is due to a modified reaction to severe hypoxia, but is not the result of Trx-1 accumulation during preconditioning.

Expression and localization of gastrin messenger RNA and peptide in spermatogenic cells.

In previous studies we have shown that the gene encoding cholecystokinin (CCK) is expressed in spermatogenic cells of several mammalian species. In the present study we show that a gene homologous to the CCK-related hormone, gastrin, is expressed in the human testis. The mRNA hybridizing to a human gastrin cDNA probe in the human testis was of the same size (0.7 kb) as gastrin mRNA in the human antrum. By in situ hybridization the gastrinlike mRNA was localized to seminiferous tubules. Immunocytochemical staining of human testis revealed gastrinlike peptides in the seminiferous tubules primarily at a position corresponding to spermatids and spermatozoa. In ejaculated spermatozoa gastrinlike immunoreactivity was localized to the acrosome. Acrosomal localization could also be shown in spermatids with electron microscopy. Extracts of the human testis contained significant amounts of progastrin, but no bioactive amidated gastrins. In contrast, ejaculated sperm contained mature carboxyamidated gastrin 34 and gastrin 17. The concentration of gastrin in ejaculated human spermatozoa varied considerably between individuals. We suggest that amidated gastrin (in humans) and CCK (in other mammals) are released during the acrosome reaction and that they may be important for fertilization.

A prospero-related homeobox gene Prox-1 is expressed during postnatal brain development as well as in the adult rodent brain.

Prox-1, a prospero-related homeobox gene, is known to be an important transcription factor during embryogenesis. However, very little is known about Prox-1 expression and functions in the adult nervous system. Here we have investigated the expression pattern of Prox-1 mRNA and protein during postnatal brain development and in adult rat and mouse brains using in situ hybridization (ISH), immunohistochemistry (IHC) and Western blotting. In the developing and adult brain, we found prominent, but restricted Prox-1 mRNA expression in the dentate gyrus of the hippocampus, in some thalamic nuclei, notably in the anterior thalamus, and in the cerebellar cortex. Other brain regions, such as the hypothalamus and nuclei belonging to the midbrain, revealed a moderate level of Prox-1 mRNA expression. In developing cerebral cortex, Prox-1 mRNA was seen only in the thin layer under the pial surface postnatally, and the signal almost disappeared by the 28th postnatal day (PD). Using IHC and ISH approaches, we demonstrated rather restricted, but intense Prox-1 labeling in adult brain of both rat and mouse species. During postnatal brain development Prox-1 proteins by IHC, were below the detection limit at PD 14, while Prox-1 mRNA remained at a high level. Western blotting demonstrated the existence of two different variants of Prox-1 protein, one of which was about 20 kDa larger than ordinary size. During the first PDs, the larger variant predominated. At PD 14, neither protein variant could be detected. From PD 16 onwards the smaller variant started to predominate and by PD 30 the larger size protein had almost disappeared. The prominent but limited distribution of Prox-1 in the brain suggests its potentially important role during postnatal brain development and in adult CNS, which remains to be ascertained in future studies.

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.

Isolation and characterization of AINT: a novel ARNT interacting protein expressed during murine embryonic development.

Basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) proteins form dimeric transcription factors to mediate diverse biological functions including xenobiotic metabolism, hypoxic response, circadian rhythm and central nervous system midline development. The Ah receptor nuclear translocator protein (ARNT) plays a central role as a common heterodimerization partner. Herein, we describe a novel, embryonically expressed, ARNT interacting protein (AINT) that may be a member of a larger coiled-coil PAS interacting protein family. The AINT C-terminus mediates interaction with the PAS domain of ARNT in yeast and interacts in vitro with ARNT and ARNT2 specifically. AINT localizes to the cytoplasm and overexpression leads to non-nuclear localization of ARNT. A dynamic pattern of AINT mRNA expression during embryogenesis and cerebellum ontogeny supports a role for AINT in development.

Expression of early gene proteins, structural changes in brain neurons in hypobaric hypoxia, and the correcting effects of preconditioning.

The Nissl method and immunocytochemistry were used to study the effects of severe hypobaric hypoxia and its actions in combination with the preconditioning actions of moderate hypoxia on the expression of the early gene proteins c-Fos and NGFI-A as well as structural changes in hippocampal and neocortical neurons in the rat brain. Severe hypoxia was found to suppress c-Fos and NGFI-A synthesis (3-24 h after exposure) and to induce delayed (days 3-7) structural damage to neurons, of the "light" and predominantly the "dark" types, which appear to reflect the development of necrotic and apoptotic processes respectively. Preconditioning with the regime used here corrected these derangements, resulting in increases in the expression of early gene proteins and significant reductions in structural damage to neurons after severe hypoxia.

Expression of diazepam binding inhibitor-like immunoreactivity in rat testis is dependent on pituitary hormones.

Diazepam binding inhibitor-like immunoreactivity (DBI-LI) in normal and hypophysectomized (HPX) rat testis was studied by light and electron microscopic immunohistochemistry. In normal testis DBI-LI was observed in interstitial Leydig cells and Sertoli cells of most seminiferous tubules. At the electron microscopic level, DBI-LI was distributed throughout the cytoplasm of labeled cells; a concentration of the labeling product was observed in the smooth endoplasmic reticulum, Golgi apparati, and the outer membrane of the mitochondria. In the seminiferous epithelium, labeled processes of Sertoli cells could be traced among developing germ cells. After HPX a gradual disappearance of DBI-LI was observed in all Leydig cells. The number of labeled Sertoli cells was reduced in the majority of tubules after HPX, whereas in some tubules the staining was only slightly reduced. Replacement therapy of the HPX animals with human CG prevented the disappearance of DBI-LI from Leydig cells, whereas replacement with FSH did not prevent the disappearance of DBI-LI in Leydig cells. In Sertoli cells replacement therapies were not effective in restoring DBI-LI. In rat testis DBI and mitochondrial benzodiazepine binding sites are highly concentrated in the Leydig cells. Mitochondrial benzodiazepine binding sites and DBI are involved in the regulation of steroid production. As the expression of DBI-LI is under the control of pituitary hormones, DBI may play a role as an endogenous regulator of intracellular metabolic functions, such as steroidogenesis, in rat testis.

Fos-like immunoreactivity in the rat hypothalamic-pituitary axis after immobilization stress.

The effect of immobilization stress on the expression of the protooncogene c-fos in the rat pituitary and hypothalamus was investigated immunohistochemically using different polyclonal antibodies raised against the c-fos protein (Fos). After a 4 h immobilization, Fos-like immunoreactivity (Fos-LI) increased substantially in the parvocellular part of the paraventricular nucleus and in the intermediate and anterior lobe of the pituitary. The majority of the Fos-immunoreactive cells in the pituitary contained corticotropin, which was demonstrated by immunohistochemical double-staining. Since the paraventricular nucleus contains a large number of glucocorticoid receptor immunoreactive cells, the effect of a synthetic glucocorticoid, dexamethasone, on the induction of Fos-LI was studied. Dexamethasone treatment before immobilization considerably reduced the stress-induced expression of Fos-LI in the anterior and intermediate lobe of the pituitary but did not alter the induction of Fos-LI in the paraventricular nucleus. The present results demonstrate that immobilization stress induces Fos-LI both in the hypothalamus and in the pituitary, suggesting that Fos may be involved in regulating the synthesis of different mediators of stress response, such as CRF- and POMC-derived peptides. Apparently glucocorticoids do not directly repress c-fos expression, since dexamethasone did not affect the induction of Fos-LI in the paraventricular nucleus. The reduction of stress-induced Fos-LI in the pituitary by dexamethasone is possibly due to the diminished release of CRF factor from the paraventricular neurons.

Stage-specific expression of pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA in the rat seminiferous tubules.

We have used in situ hybridization and Northern blot analysis with oligonucleotide probe to characterize the site of pituitary adenylate cyclase-activating polypeptide (PACAP) synthesis in the rat testis. We observed strong hybridization signal in one third of the cross-sections of the seminiferous tubules, whereas some tubules were devoid of hybridization signal, thus suggesting that PACAP mRNA is expressed in a stage-specific manner. More detailed analysis showed that PACAP mRNA was present in round spermatids at stages III-VII of the cycle. Northern blot hybridization to RNAs extracted from samples of seminiferous tubules at different stages of the epithelial cycle confirmed that expression of PACAP mRNA is restricted to specific stages of the cycle. The highest amount of PACAP mRNA was detected at stages V to early -VII of the cycle, whereas very low levels of mRNA were present at stages I-II and IX-XIV. The present results demonstrate that PACAP mRNA is expressed in the developing germ cells. This suggests that PACAP may function as a paracrine or autocrine regulatory factor for the Sertoli and germ cells, with a specific function during early spermiogenesis, shortly before the onset of nuclear elongation, at the last period of haploid gene activity.

Expression of peroxisome proliferator-activated receptor alpha messenger ribonucleic acid and protein in human and rat testis.

Peroxisome proliferator-activated receptor a (PPARalpha), a member of the steroid hormone receptor superfamily, has been linked to lipid homeostasis and tumorigenesis in tissues with high expression of receptor protein. On the other hand, the role of PPARalpha in tissues with a lower expression is not well known. Here we demonstrate the localization of PPARalpha messenger RNA (mRNA) and protein in developing and adult rat testis. Additionally, we demonstrate the expression of PPARalpha protein in adult human testis. Our experiments with Northern analysis, in situ hybridization and immunocytochemistry reveal a complex distribution of PPARalpha in tubular and interstitial cells of both adult and developing rat testis. The overall expression is rather low but may be modified by exogenous or endogenous stimuli. An up-regulation of PPARalpha mRNA could be observed after stimulation with FSH. In the developing rat testis, a clear expression of PPARalpha mRNA was present from the first days after birth. Additionally, PPARalpha mRNA and protein increased toward adulthood. In adult human testis PPARalpha immunoreactivity (IR) was present in interstitial Leydig cells and tubular cells. In the seminiferous epithelium of adult human testis the expression of PPARalpha-IR could be seen in meiotic spermatocytes, spermatids and myoid peritubular cells. The findings of our study suggest that PPARalpha may be involved in the regulation of growth and differentiation of tubular and interstitial cells in rat and human testis.

Human estrogen receptor beta-gene structure, chromosomal localization, and expression pattern.

The estrogen receptor (ER) is a ligand-activated transcription factor that mediates the effects of the steroid hormone 17 beta-estradiol, in both males and females. Since the isolation and cloning of ER, the consensus has been that only one such receptor exists. The finding of a second subtype of ER (ER beta) has caused considerable excitement amongst endocrinologists. In this article, we present data regarding the genomic structure and chromosomal localization of the human ER beta gene, demonstrating that two independent ER genes do exist in the human. Furthermore, we present data regarding the tissue distribution of human ER beta, showing that this receptor is expressed in multiple tissues. For instance, ER beta is found in developing spermatids of the testis, a finding of potential relevance for the ongoing debate on the effects of environmental estrogens on sperm counts. In addition, we find ER beta in ovarian granulosa cells, indicating that estrogens also participate in the regulation of follicular growth in the human.

TACC3 expression is tightly regulated during early differentiation.

Transforming acidic coiled-coil (TACC) proteins are hypothesized to play a role in normal cellular growth and differentiation and to be involved in centrosomal microtubule stabilization. Our current studies aim to delineate the expression pattern of TACC3 protein during cellular differentiation and in a variety of normal human tissues. TACC3 is known to be upregulated in differentiating erythroid progenitor cells following treatment with erythropoietin and is required for replication of hematopoietic stem cells. However, we demonstrate that a dramatic upregulation of TACC3 also occurs during the early differentiation of NIH 3T3-L1 cells into adipocytes and PC12 cells into neurons, indicating that TACC3 mediates cellular differentiation in several cell types. Using real-time PCR, we quantitated the mRNA levels of TACC3 compared to TACC1 and TACC2 in various human adult tissues. We observed the highest expression of TACC3 mRNA in testis, spleen, thymus and peripheral blood leukocytes, all tissues undergoing high rates of differentiation, and a lower level of expression in ovary, prostate, pancreas, colon, small intestine, liver and kidney. In contrast, TACC1 and TACC2 mRNA levels are more widespread. By immunohistochemistry, we confirm that the TACC3 protein localizes to differentiating cell types, including spermatocytes, oocytes, epithelial cells, bone marrow cells and lymphocytes. Thus, these observations are concordant with a basic role for TACC3 during early stages of differentiation in normal tissues.

Decreased mRNA levels for exocytotic proteins in the pituitary of aged rats.

Several protein components that are involved in the molecular regulation of transmitter release have been identified in neuronal, neuroendocrine and endocrine tissues. The expression of VAMP-2 (vesicle-associated membrane protein), munc-18 (mammalian homologue of unc-18) and SNAP-25 (synaptosomal-associated protein of 25 kDa) mRNA was studied in the rat anterior and intermediate pituitary gland of adult (2 months) and old (24 months) rats using in situ hybridization. In the pituitary anterior lobe of aged rats, there was a significant decrease in VAMP-2 (33%), munc-18 (17%) and SNAP-25 (20%) mRNA as compared to adult rats. In the intermediate lobe, there was a significant decrease in VAMP-2 (48%) and SNAP-25 (32%) mRNA of aged rats, whereas munc-18 mRNA levels were not significantly changed. Pituitaries from aged rats showed an increase in size which was paralleled by a significant decrease in the number of cells per unit area in the intermediate lobe, whereas the number was unaltered in the anterior lobe. The results suggest a genuine decrease in mRNA for exocytotic protein mRNA in the anterior pituitary, but that part of the decrease in the expression of VAMP-2 and SNAP-25 mRNA in the intermediate lobe can be explained by a decreased number of cells per unit area. The decline in anterior pituitary hormone secretion reported in aged rats appears to be parallelled by a down-regulation in mRNA levels for several proteins involved in the molecular regulation of exocytosis.