Direct diffusion of anti-Müllerian hormone from both the cranial and caudal regions of the testis during early gonadal development in mice.

BACKGROUND: The Müllerian duct (MD), the primordium of the female reproductive tract, is also formed in males during the early stage of development, then regresses due to the anti-Müllerian hormone (AMH) secreted from the testes. However, the detailed diffusion pathway of AMH remains unclear. We herein investigated the mechanism by which AMH reaches the middle region of the MD using an organ culture system. RESULTS: Injection of recombinant human AMH into the testis around the start of MD regression induced diffuse immunoreactivity in the mesonephros near the injection site. When the testis and mesonephros were cultured separately, the diameters of both cranial and middle MDs were significantly increased compared to the control. In the testis-mesonephros complex cultured by inhibiting the diffusion of AMH through the cranial region, the cranial MD diameter was significantly increased compared to the control, and there was no difference in middle MD diameter. CONCLUSIONS: These results indicate that AMH, which infiltrates from the testis through the cranial region at physiological concentrations, induces regression of the cranial MD at the start of MD regression. They also indicate that AMH infiltrating through the caudal regions induces regression of the middle MD.

Hydrogen gas can ameliorate seizure burden during therapeutic hypothermia in asphyxiated newborn piglets.

BACKGROUND: We previously reported that hydrogen (H2) gas combined with therapeutic hypothermia (TH) improved short-term neurological outcomes in asphyxiated piglets. However, the effect on seizure burden was unclear. Using amplitude-integrated electroencephalography (aEEG), we compared TH + H2 with TH alone in piglets 24 h after hypoxic-ischemic (HI) insult. METHODS: After a 40-min insult and resuscitation, 36 piglets ≤24 h old were divided into three groups: normothermia (NT, n = 14), TH alone (33.5 ± 0.5 °C, 24 h, n = 13), and TH + H2 (2.1-2.7% H2 gas, 24 h, n = 9). aEEG was recorded for 24 h post-insult and its background pattern, status epilepticus (SE; recurrent seizures lasting >5 min), and seizure occurrence (Sz; occurring at least once but not fitting the definition of SE) were evaluated. Background findings with a continuous low voltage and burst suppression were considered abnormal. RESULTS: The percentage of piglets with an abnormal aEEG background (aEEG-BG), abnormal aEEG-BG+Sz and SE was lower with TH + H2 than with TH at 24 h after HI insult. The duration of SE was shorter with TH + H2 and significantly shorter than with NT. CONCLUSIONS: H2 gas combined with TH ameliorated seizure burden 24 h after HI insult. IMPACT: In this asphyxiated piglet model, there was a high percentage of animals with an abnormal amplitude-integrated electroencephalography background (aEEG-BG) after hypoxic-ischemic (HI) insult, which may correspond to moderate and severe hypoxic-ischemic encephalopathy (HIE). Therapeutic hypothermia (TH) was associated with a low percentage of piglets with EEG abnormalities up to 6 h after HI insult but this percentage increased greatly after 12 h, and TH was not effective in attenuating seizure development. H2 gas combined with TH was associated with a low percentage of piglets with an abnormal aEEG-BG and with a shorter duration of status epilepticus at 24 h after HI insult.

Maternal separation early in life induces excessive activity of the central amygdala related to abnormal aggression.

Epidemiological studies have indicated that child maltreatment, such as neglect, is a risk factor of escalated aggression, potentially leading to delinquency and violent crime in the future. However, little is known about the mechanisms by which an early adverse environment may later cause violent behavior. In this study, we aimed to thoroughly examine the association between aggression against conspecific animals and the activity of amygdala subnuclei using the maternal separation (MS) model, which is a common model of early life stress. In the MS group, pups of Sprague-Dawley rats were separated from their dam during postnatal days 2-20 (twice a day, 3 h each). We only included 9-week-old male offspring for each analysis and compared the MS group with the mother-reared control group; both groups were raised by the same dam during postnatal days 2-20. The results revealed that the MS group exhibited higher aggression and excessive activity of only the central amygdala (CeA) among the amygdala subnuclei during the aggressive behavior test. Moreover, a significant positive correlation was observed between higher aggression and CeA activation. While CeA activity is known to be involved in hunting behavior for prey, some previous studies have also indicated a relationship between CeA and intraspecific aggression. It remains unclear, however, whether excessive CeA activity directly induces intraspecific aggression. Therefore, we stimulated the CeA using optogenetics with 8-week-old rats to clarify the relationship between intraspecific aggression and CeA activity. Notably, CeA activation resulted in higher aggression, even when the opponent was a conspecific animal. In particular, bilateral CeA activation resulted in more severe displays of aggressive behavior than necessary, such as biting a surrendered opponent. These findings suggest that an adverse environment during early development intensifies aggression through excessive CeA activation, which can increase the risk of escalating to violent behavior in the future.

Genetic and histopathological analysis of spermatogenesis after short-term testicular torsion in rats.

BACKGROUND: Patients with testicular torsion (TT) may exhibit impaired spermatogenesis from reperfusion injury after detorsion surgery. Alteration in the expressions of spermatogenesis-related genes induced by TT have not been fully elucidated. METHODS: Eight-week-old Sprague-Dawley rats were grouped as follows: group 1 (sham-operated), group 2 (TT without reperfusion) and group 3 (TT with reperfusion). TT was induced by rotating the left testis 720° for 1 h. Testicular reperfusion proceeded for 24 h. Histopathological examination, oxidative stress biomarker measurements, RNA sequencing and RT-PCR were performed. RESULTS: Testicular ischemia/reperfusion injury induced marked histopathological changes. Germ cell apoptosis was significantly increased in group 3 compared with group 1 and 2 (mean apoptotic index: 26.22 vs. 0.64 and 0.56; p = 0.024, and p = 0.024, respectively). Johnsen score in group 3 was smaller than that in group 1 and 2 (mean: 8.81 vs 9.45 and 9.47 points/tubule; p = 0.001, p < 0.001, respectively). Testicular ischemia/reperfusion injury significantly upregulated the expression of genes associated with apoptosis and antioxidant enzymes and significantly downregulated the expression of genes associated with spermatogenesis. CONCLUSION: One hour of TT followed by reperfusion injury caused histopathological testicular damage. The relatively high Johnsen score indicated spermatogenesis was maintained. Genes associated with spermatogenesis were downregulated in the TT rat model. IMPACT: How ischemia/reperfusion injury in testicular torsion (TT) affects the expressions of genes associated with spermatogenesis has not been fully elucidated. This is the first study to report comprehensive gene expression profiles using next generation sequencing for an animal model of TT. Our results revealed that ischemia/reperfusion injury downregulated the expression of genes associated with spermatogenesis and sperm function in addition to histopathological damage, even though the duration of ischemia was short.

Hypothermia cannot ameliorate renal fibrosis after asphyxia in the newborn piglet.

BACKGROUND: The effects of therapeutic hypothermia (TH) on renal function are not widely reported, especially in longer term animal models. The hypothesis of this study was that TH of the kidneys of hypoxic-ischemic newborn piglets would reduce pathological renal fibrosis. METHODS: Twenty-five newborn piglets obtained within 24 h of birth were classified into a control group (n = 5), an hypoxic insult with normothermia (HI-NT) group (n = 12), and an hypoxic insult with TH (HI-TH) group (33.5 °C ± 0.5 °C for 24 h; n = 8). Five days after the insult, all piglets were sacrificed under deep anesthesia by isoflurane inhalation. The kidneys were perfused with phosphate-buffered paraformaldehyde and immersed in formalin buffer. Territory fibrosis was studied and scored in the renal medulla using Azan staining. RESULTS: Fibrosis area scores (means ± standard deviations) based on Azan staining were 1.00 ± 0.46 in the control group, 2.85 ± 0.93 in the HI-NT group, and 3.58 ± 1.14 in the HI-TH group. The fibrosis area of the HI-NT and HI-TH groups was larger than that of the control. The HI-NT and HI-TH groups were not statistically different. CONCLUSIONS: Renal fibrosis is affected by perinatal asphyxia and cannot be prevented by TH, based on histopathological findings.

Accumulation of microdamage and low bone mass in the femoral head as a cause of subchondral insufficiency fracture in a patient with osteogenesis imperfecta.

Subchondral insufficiency fractures of the femoral head are generally considered to be osteoporosis-related fragility fractures. There have been reports of microfractures being found in subchondral bone on pathological examination. However, the mechanism of these microfractures is not known. In this report, we describe a patient with osteogenesis imperfecta who developed a subchondral insufficiency fracture of the femoral head after a fall that had resulted in a subcapital femoral neck fracture. Bipolar hemiarthroplasty was performed, and bone at the femoral head and neck was sampled for pathophysiological examination. Hematoxylin and eosin staining revealed microfractures and microcallus in the subchondral bone in the femoral head, indicating healing of a subchondral insufficiency fracture before the subcapital femoral neck fracture. Moreover, decreased bone volume and accumulated microdamage were observed in the subchondral bone but not in the cancellous bone in the femoral neck. These findings suggest that subchondral insufficiency fracture of the femoral head is a stress fracture caused by accumulation of microdamage in fragile subchondral bone.

Accumulation of microdamage in subchondral bone at the femoral head in patients with end-stage osteoarthritis of the hip.

In end-stage osteoarthritis (OA) of the hip, the effect of bone metabolism with and without cartilage is unclear. In this study, we aimed to investigate histomorphology and microdamage in the subchondral bone of the femoral head in areas with and without articular cartilage in patients with end-stage OA. Nineteen femoral heads were evaluated in 10 women who underwent total hip arthroplasty for OA and in nine cadaveric controls (CNT). Chondral thickness and subchondral bone plate thickness (SBP.Th) were measured in 5-mm-wide areas where cartilage was lost (area A) or preserved (area B) in OA and in corresponding areas in the load-bearing portion of the femoral head in the CNT. Histomorphometry and microdamage in 5 × 5-mm areas of cancellous bone were assessed. SBP.Th and bone volume were significantly greater in area A than in area B or in the CNT. Osteoid volume was significantly greater in area A than in area B or in the CNT. There was no significant difference in eroded surface between area A and CNT. Microcrack density was significantly greater in area A than in area B or in the CNT. Although accumulation of microdamage was caused by concentration of stress on the subchondral bone in the cartilage loss area in end-stage OA, remodeling for microdamage repairing mechanism was not enhanced. It was considered that the subchondral cancellous bone volume was increased because of modeling, not remodeling, by stress concentration due to articular cartilage loss.

Corticotropin-releasing hormone-binding protein is up-regulated by brain-derived neurotrophic factor and is secreted in an activity-dependent manner in rat cerebral cortical neurons.

A recent study revealed that corticotropin-releasing hormone (CRH) in the cerebral cortex (CTX) plays a regulatory role in emotional behaviors in rodents. Given the functional interaction between brain-derived neurotrophic factor (BDNF) and the CRH-signaling pathway in the hypothalamic-pituitary-adrenal axis, we hypothesized that BDNF may regulate gene expression of CRH and its related molecules in the CTX. Findings of real-time quantitative PCR (RT-qPCR) indicated that stimulation of cultured rat cortical neurons with BDNF led to marked elevations in the mRNA levels of CRH and CRH-binding protein (CRH-BP). The BDNF-induced up-regulation of CRH-BP mRNA was attenuated by inhibitors of tropomyosin related kinase (Trk) and MEK, but not by an inhibitor for PI3K and Phospholipase C gamma (PLCγ). The up-regulation was partially blocked by an inhibitor of lysine-specific demethylase (KDM) 6B. Fluorescent imaging identified the vesicular pattern of pH-sensitive green fluorescent protein-fused CRH-BP (CRH-BP-pHluorin), which co-localized with mCherry-tagged BDNF in cortical neurons. In addition, live-cell imaging detected drastic increases of pHluorin fluorescence in neurites upon membrane depolarization. Finally, we confirmed that tetrodotoxin partially attenuated the BDNF-induced up-regulation of CRH-BP mRNA, but not that of the protein. These observations indicate the following: In cortical neurons, BDNF led to gene expression of CRH-BP and CRH. TrkB, MEK, presumably ERK, and KDM6B are involved in the BDNF-induced gene expression of CRH-BP, and BDNF is able to induce the up-regulation in a neuronal activity-independent manner. It is suggested that CRH-BP is stored into BDNF-containing secretory granules in cortical neurons, and is secreted in response to membrane depolarization.

Perfusion with carbon monoxide does not affect extracellular glutamate in dialysates of the hippocampus of freely moving mice.

Carbon monoxide (CO) produces several neurological effects, including cognitive, mood, and behavioral disturbance. Glutamate is thought to play a particularly important role in learning and memory. Thus, the present study was aimed at investigating the local effect of CO on the glutamate level in the hippocampus of mice using in vivo reverse microdialysis. Mice were perfused with Ringer's solution (control) or CO (60-125 µM) in Ringer's solution into the hippocampus via microdialysis probe. Dialysate samples were collected every 20 min, and then analyzed with high-performance liquid chromatography coupled to an electrochemical detector. The result revealed that the perfusion with CO had no significant effect on glutamate levels (p = 0.316) as compared to the control group. This finding does not support a local CO rise as the cause of the increased glutamate level in the hippocampus of mice.

[A Case of Moyamoya Disease with Postoperative Cerebral Hyperperfusion Syndrome Followed by Cerebral Infarction due to Watershed Shift].

The concept of "watershed shift"(WS)has been proposed as a cause of the ischemic complications following a superficial temporal artery-middle cerebral artery(STA-MCA)bypass operation performed for the management of moyamoya disease. Previous reports have observed that only 1.2-5.7% of the patients who underwent a bypass operation for the management of moyamoya disease developed cerebral infarction secondary to the WS phenomenon. To date, the WS phenomenon has not been objectively proven on imaging studies. We describe a 39-year-old woman who presented with right facial palsy and aphasia. Magnetic resonance imaging revealed cerebral infarction in the left frontal lobe secondary to moyamoya disease. Three days after undergoing the left STA-MCA bypass procedure, she showed deterioration in aphasia secondary to the occurrence of cerebral hyperperfusion syndrome(CHPS). Diffusion-weighted imaging(DWI)performed on postoperative day(POD)1 and 5 showed no area of high signal intensity. DWI performed on POD 8 showed an area of high signal intensity in the deep white matter of the left parietal lobe outside the range of the craniotomy. Postoperative fusion images of computed tomography angiography and DWI performed on POD 8 showed that the blood flow through the MCA from the bypass graft and that through the posterior cerebral artery crossed each other at the surface of the subcortical infarction. In the present case, the WS could be directly confirmed on imaging studies, and the cerebral infarction may have occurred secondary to WS concomitant with CHPS. Clinicians need to be aware of the WS phenomenon even after performing a direct bypass to treat adults with moyamoya disease.

Prolonged maternal separation attenuates BDNF-ERK signaling correlated with spine formation in the hippocampus during early brain development.

Maternal separation (MS) is known to affect hippocampal function such as learning and memory, yet the molecular mechanism remains unknown. We hypothesized that these impairments are attributed to abnormities of neural circuit formation by MS, and focused on brain-derived neurotrophic factor (BDNF) as key factor because BDNF signaling has an essential role in synapse formation during early brain development. Using rat offspring exposed to MS for 6 h/day during postnatal days (PD) 2-20, we estimated BDNF signaling in the hippocampus during brain development. Our results show that MS attenuated BDNF expression and activation of extracellular signal-regulated kinase (ERK) around PD 7. Moreover, plasticity-related immediate early genes, which are transcriptionally regulated by BDNF-ERK signaling, were also reduced by MS around PD 7. Interestingly, detailed analysis revealed that MS particularly reduced expression of BDNF gene and immediate early genes in the cornu ammonis 1 (CA1) of hippocampus at PD 7. Considering that BDNF-ERK signaling is involved in spine formation, we next evaluated spine formation in the hippocampus during the weaning period. Our results show that MS particularly reduced mature spine density in proximal apical dendrites of CA1 pyramidal neurons at PD 21. These results suggest that MS could attenuate BDNF-ERK signaling during primary synaptogenesis with a region-specific manner, which is likely to lead to decreased spine formation and maturation observed in the hippocampal CA1 region. It is speculated that this incomplete spine formation during early brain development has an influence on learning capabilities throughout adulthood.

Short-term ethanol exposure causes imbalanced neurotrophic factor allocation in the basal forebrain cholinergic system: a novel insight into understanding the initial processes of alcohol addiction.

Alcohol ingestion affects both motor and cognitive functions. One brain system that is influenced by ethanol is the basal forebrain (BF) cholinergic projection system, which projects to diverse neocortical and limbic areas. The BF is associated with memory and cognitive function. Our primary interest is the examination of how regions that receive BF cholinergic projections are influenced by short-term ethanol exposure through alterations in the mRNA levels of neurotrophic factors [nerve growth factor/TrkA, brain-derived neurotrophic factor/TrkB, and glial-derived neurotrophic factor (GDNF)/GDNF family receptor α1]. Male BALB/C mice were fed a liquid diet containing 5 % (v/v) ethanol. Pair-fed control mice were maintained on an identical liquid diet, except that the ethanol was isocalorically substituted with sucrose. Mice exhibiting signs of ethanol intoxication (stages 1-2) were used for real-time reverse transcription-polymerase chain reaction analyses. Among the BF cholinergic projection regions, decreased levels of GDNF mRNA and increased levels of TrkB mRNA were observed in the basal nucleus, and increased levels of TrkB mRNA were observed in the cerebral cortex. There were no significant alterations in the levels of expression of relevant neurotrophic factors in the septal nucleus and hippocampus. Given that neurotrophic factors function in retrograde/anterograde or autocrine/paracrine mechanisms and that BF cholinergic projection regions are neuroanatomically connected, these findings suggested that an imbalanced allocation of neurotrophic factor ligands and receptors is an initial phenomenon in alcohol addiction. The exact mechanisms underlying this phenomenon in the BF cholinergic system are unknown. However, our results provide a novel notion for the understanding of the initial processes in alcohol addiction.

Establishment of an organ culture system to maintain the structure of mouse Müllerian ducts during development.

We previously showed that the anti-Müllerian hormone (AMH), infiltrating from the testis to the mesonephros reaches the cranial and middle regions of the Müllerian duct (MD) and induces their regression using an organ culture in mice. However, it is difficult to maintain structural integrity, such as the length and diameter and normal direction of elongation of the caudal region of the MD, in conventional organ culture systems. Therefore, the pathway of AMH to the caudal MD region remains uncharted. In this study, we established an organ culture method that can maintain the morphology of the caudal region of the MD. The gonad-mesonephros complex, metanephros, and urinary bladder of mouse fetuses at 12.5 dpc attached to the body trunk were cultured on agarose gels for 72 hr. The cultured caudal region of the mesonephros was elongated along the body trunk, and the course of the mesonephros was maintained in many individuals. In males, mesenchymal cells aggregated around the MD after culture. Moreover, the male MD diameter was significantly smaller than the female. Based on these results, it was concluded that the development of the MD was maintained in the present organ culture system. Using this culture system, AMH infiltration to the caudal region of the MD can be examined without the influence of AMH in the blood. This culture system is useful for clarifying the regression mechanism of the caudal region of the MD.

Early postnatal maternal separation causes alterations in the expression of ß3-adrenergic receptor in rat adipose tissue suggesting long-term influence on obesity.

The effects of early postnatal maternal deprivation on the biological characteristics of the adipose tissue later in life were investigated in the present study. Sprague-Dawley rats were classified as either maternal deprivation (MD) or mother-reared control (MRC) groups. MD was achieved by separating the rat pups from their mothers for 3h each day during the 10-15 postnatal days. mRNA levels of mitochondrial uncoupling protein 1 (UCP-1), ß3-adrenergic receptor (ß3-AR), and prohibitin (PHB) in the brown and white adipose tissue were determined using real-time RT-PCR analysis. UCP-1, which is mediated through ß3-AR, is closely involved in the energy metabolism and expenditure. PHB is highly expressed in the proliferating tissues/cells. At 10 weeks of age, the body weight of the MRC and MD rats was similar. However, the levels of the key molecules in the adipose tissue were substantially altered. There was a significant increase in the expression of PHB mRNA in the white adipose tissue, while the ß3-AR mRNA expression decreased significantly, and the UCP-1 mRNA expression remained unchanged in the brown adipose tissue. Given that these molecules influence the mitochondrial metabolism, our study indicates that early postnatal maternal deprivation can influence the fate of adipose tissue proliferation, presumably leading to obesity later in life.

The effect of blood transfusion on cerebral hemodynamics in preterm infants.

BACKGROUND: Anemia of prematurity commonly occurs in infants with very low birth weight; blood transfusion is an important treatment. However, there is no clear evidence to support the criteria currently widely used, based on blood hemoglobin (bHb) and hematocrit indices. Previous studies showed that overtransfusion or a low threshold for transfusion could induce complications or neurologic sequelae, respectively. We hypothesized that a cerebral hemodynamic index may provide an appropriate criterion for determining the need for transfusion in anemic preterm infants. STUDY DESIGN AND METHODS: We used near-infrared time-resolved spectroscopy to measure cerebral hemoglobin oxygen saturation (ScO2 ) and cerebral blood volume (CBV) before and after transfusion in 19 infants (24 measurements) with anemia of prematurity. The median gestational age was 27 weeks 0 days, median birth weight was 751 g, and median postconceptual age at transfusion was 30 weeks 4 days. RESULTS: bHb levels before and after transfusion (mean ± SD) were 9.3 ± 1.4 and 13.7 ± 1.3 g/dL, respectively. After transfusion, CBV significantly decreased from 2.63 ± 0.60 to 2.13 ± 0.26 mL/100 g of brain, and ScO2 significantly increased from 72.8 ± 4.3% to 74.7 ± 4.2%. CONCLUSION: After transfusion, CBV changes were significantly greater with low compared to high pretransfusion Hb levels. This reflected the physiologic response to severe anemia in premature infants, which is to increase CBV and decrease ScO2 . Therefore, CBV and ScO2 may be useful markers for determining the need for transfusion in very-low-birth-weight infants.

Low-dose nicotine facilitates spatial memory in ApoE-knockout mice in the radial arm maze.

Here, we investigated the effects of nicotine on spatial memory in ApoE-knockout (ApoE-KO) and wild-type (WT) mice in a radial arm maze. Training occurred on three consecutive days and the test was performed on day 4, with one trial per day. Then on day 4, animals were administered nicotine (0.1, 0.25, 0.5, and 1.0 mg/kg) or the antagonist of nicotinic receptors (nAChRs) mecamylamine (MEC 2 mg/kg) alone or together with 0.1 mg/kg nicotine. The number of errors in the first eight choices was recorded. The results were that 0.1 mg/kg nicotine decreased errors in ApoE-KO mice, while 0.1 and 0.25 mg/kg nicotine reduced errors in WT mice, indicating that lower doses of nicotine elicit a memory improvement. In contrast, 1.0 mg/kg nicotine increased errors in WT mice, but not in ApoE-KO mice. MEC alone had no noticeable effect on errors in either strain of mice. However, co-administration of 0.1 mg/kg nicotine and MEC increased errors and reduced the effects of nicotine in WT mice, but not in ApoE-KO mice. Our study found a biphasic effect of nicotine in WT mice: it improves spatial memory at lower doses and impairs it at a higher dose. In ApoE-KO mice, nicotine improves memory at a low dose and has no effect at a higher dose, suggesting that the ApoE deficiency may influence the efficacy of nicotine. Moreover, a reversal of nicotinic effects with MEC was seen in WT mice, indicating the likelihood of the involvement of nAChRs in the spatial-memory response to nicotine.

Substrain differences in Sry expression at the stage of sex determination in C57BL/6 mouse strains.

The expression of sex determining region of the Y chromosome (Sry) in the fetal gonads is important for male development. In a mouse model of disorders of sex development (C57BL/6 (B6)-XYPOS), the gonadal phenotype and the timing of Sry expression differ due to differences among B6 substrains as the genetic background. Since differences in Sry expression among B6 substrains have been speculated, the present study examined Sry expression in B6J, B6JJmsSlc, and B6NCrl mice. These substrains differed in the number of Sry-expressing cells in the gonads of embryonic mice at each developmental stage, with B6NCrl having more than the other strains. The substrains differed also in the number of Sry-expressing cells between the left and right gonads, with B6J and B6NCrl, but not B6JJmsSlc, showing left gonad-dominant Sry expression. Substrain differences existed also in the distribution of Sry-expressing cells in the medial and lateral directions of gonads. In addition, in the left gonad-dominant Sry-expressing substrains B6J and B6NCrl, the medial and central regions of the left gonad had more Sry-expressing cells than those of the right gonad. Substrains of B6 mice have not always been considered in sex differentiation studies. In the present study, however, we observed substrain differences in the number of Sry-expressing cells, left-right distribution, and medial/lateral distribution during the early stages of gonadal development in B6 mice. Therefore, future studies on sex differentiation in B6 mice should consider substrain differences.

Impact of hydrogen gas inhalation during therapeutic hypothermia on cerebral hemodynamics and oxygenation in the asphyxiated piglet.

We previously reported the neuroprotective potential of combined hydrogen (H2) gas ventilation therapy and therapeutic hypothermia (TH) by assessing the short-term neurological outcomes and histological findings of 5-day neonatal hypoxic-ischemic (HI) encephalopathy piglets. However, the effects of H2 gas on cerebral circulation and oxygen metabolism and on prognosis were unknown. Here, we used near-infrared time-resolved spectroscopy to compare combined H2 gas ventilation and TH with TH alone. Piglets were divided into three groups: HI insult with normothermia (NT, n = 10), HI insult with hypothermia (TH, 33.5 ± 0.5 °C, n = 8), and HI insult with hypothermia plus H2 ventilation (TH + H2, 2.1-2.7%, n = 8). H2 ventilation and TH were administered and the cerebral blood volume (CBV) and cerebral hemoglobin oxygen saturation (ScO2) were recorded for 24 h after the insult. CBV was significantly higher at 24 h after the insult in the TH + H2 group than in the other groups. ScO2 was significantly lower throughout the 24 h after the insult in the TH + H2 group than in the NT group. In conclusion, combined H2 gas ventilation and TH increased CBV and decreased ScO2, which may reflect elevated cerebral blood flow to meet greater oxygen demand for the surviving neurons, compared with TH alone.

Conflicting findings on the effectiveness of hydrogen therapy for ameliorating vascular leakage in a 5-day post hypoxic-ischemic survival piglet model.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a major cause of morbidity and mortality in newborns in both high- and low-income countries. The important determinants of its pathophysiology are neural cells and vascular components. In neonatal HIE, increased vascular permeability due to damage to the blood-brain barrier is associated with seizures and poor outcomes in both translational and clinical studies. In our previous studies, hydrogen gas (H2) improved the neurological outcome of HIE and ameliorated the cell death. In this study, we used albumin immunohistochemistry to assess if H2 inhalation effectively reduced the cerebral vascular leakage. Of 33 piglets subjected to a hypoxic-ischemic insult, 26 piglets were ultimately analyzed. After the insult, the piglets were grouped into normothermia (NT), H2 ventilation (H2), therapeutic hypothermia (TH), and H2 combined with TH (H2-TH) groups. The ratio of albumin stained to unstained areas was analyzed and found to be lower in the H2 group than in the other groups, although the difference was not statistically significant. In this study, H2 therapy did not significantly improve albumin leakage despite the histological images suggesting signs of improvement. Further investigations are warranted to study the efficacy of H2 gas for vascular leakage in neonatal HIE.

An enriched environment ameliorates the reduction of parvalbumin-positive interneurons in the medial prefrontal cortex caused by maternal separation early in life.

Early child maltreatment, such as child abuse and neglect, is well known to affect the development of social skills. However, the mechanisms by which such an adverse environment interrupts the development of social skills remain unelucidated. Identifying the period and brain regions that are susceptible to adverse environments can lead to appropriate developmental care later in life. We recently reported an excitatory/inhibitory imbalance and low activity during social behavior in the medial prefrontal cortex (mPFC) of the maternal separation (MS) animal model of early life neglect after maturation. Based on these results, in the present study, we investigated how MS disturbs factors related to excitatory and inhibitory neurons in the mPFC until the critical period of mPFC development. Additionally, we evaluated whether the effects of MS could be recovered in an enriched environment after MS exposure. Rat pups were separated from their dams on postnatal days (PDs) 2-20 (twice daily, 3 h each) and compared with the mother-reared control (MRC) group. Gene expression analysis revealed that various factors related to excitatory and inhibitory neurons were transiently disturbed in the mPFC during MS. A similar tendency was found in the sensory cortex; however, decreased parvalbumin (PV) expression persisted until PD 35 only in the mPFC. Moreover, the number of PV+ interneurons decreased in the ventromedial prefrontal cortex (vmPFC) on PD 35 in the MS group. Additionally, perineural net formation surrounding PV+ interneurons, which is an indicator of maturity and critical period closure, was unchanged, indicating that the decreased PV+ interneurons were not simply attributable to developmental delay. This reduction of PV+ interneurons improved to the level observed in the MRC group by the enriched environment from PD 21 after the MS period. These results suggest that an early adverse environment disturbs the development of the mPFC but that these abnormalities allow room for recovery depending on the subsequent environment. Considering that PV+ interneurons in the mPFC play an important role in social skills such as empathy, an early rearing environment is likely a very important factor in the subsequent acquisition of social skills.