Comparison of Neuronal Death, Blood-Brain Barrier Leakage and Inflammatory Cytokine Expression in the Hippocampal CA1 Region Following Mild and Severe Transient Forebrain Ischemia in Gerbils.

Transient ischemia in the brain causes blood-brain barrier (BBB) breakdown and dysfunction, which is related to ischemia-induced neuronal damage. Leakage of plasma proteins following transient ischemia is one of the indicators that is used to determine the extent of BBB dysfunction. In this study, neuronal damage/death, leakage of albumin and IgG, microgliosis, and inflammatory cytokine expression were examined in the hippocampal CA1 region, which is vulnerable to transient ischemia, following 5-min (mild) and 15-min (severe) ischemia in gerbils induced by transient common carotid arteries occlusion (tCCAo). tCCAo-induced neuronal damage/death occurred earlier and was more severe after 15-min tCCAo vs. after 5-min tCCAo. Significant albumin and IgG leakage (albumin and IgG immunoreactivity) took 1 or 2 days to begin, and immunoreactivity was markedly increased 5 days after 5-min tCCAo. While, albumin and IgG leakage began to increase 6 h after 15-min tCCAo and remained significantly higher over time than that seen in 5-min tCCAo. IgG immunoreactivity was observed in degenerating neurons and activated microglia after tCCAo, and microglia were activated to a greater extent after 15-min tCCAo than 5-min tCCAo. In addition, following 15-min tCCAo, pro-inflammatory cytokines [tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1ß)] immunoreactivity was significantly higher than that seen following 5-min tCCAo, whereas immunoreactivity of anti-inflammatory cytokines (IL-4 and IL-13) was lower in 15-min than 5-min tCCAo. These results indicate that duration of tCCAo differentially affects the timing and degree of neuronal damage or loss, albumin and IgG leakage and inflammatory cytokine expression in brain tissue. In addition, more severe BBB leakage is closely related to acceleration of neuronal damage through increased microglial activation and pro-inflammatory cytokine expression in the ischemic hippocampal CA1 region.

Ischemia-Reperfusion under Hyperthermia Increases Heme Oxygenase-1 in Pyramidal Neurons and Astrocytes with Accelerating Neuronal Loss in Gerbil Hippocampus.

It has been studied that the damage or death of neurons in the hippocampus is different according to hippocampal subregions, cornu ammonis 1-3 (CA1-3), after transient ischemia in the forebrain, showing that pyramidal neurons located in the subfield CA1 (CA1) are most vulnerable to this ischemia. Hyperthermia is a proven risk factor for brain ischemia and can develop more severe and extensive brain damage related with mortality rate. It is well known that heme oxygenase-1 (HO-1) activity and expression is increased by various stimuli in the brain, including hyperthermia. HO-1 can be either protective or deleterious in the central nervous system, and its roles depend on the expression levels of enzymes. In this study, we investigated the effects of hyperthermia during ischemia on HO-1 expression and neuronal damage/death in the hippocampus to examine the relationship between HO-1 and neuronal damage/death following 5-min transient ischemia in the forebrain using gerbils. Gerbils were assigned to four groups: (1) sham-operated gerbils with normothermia (Normo + sham group); (2) ischemia-operated gerbils with normothermia (Normo + ischemia group); (3) sham-operated gerbils with hyperthermia (39.5 ± 0.2 °C) during ischemia (Hyper + sham group); and (4) ischemia-operated gerbils with hyperthermia during ischemia (Hyper + ischemia group). HO-1 expression levels in CA1-3 of the Hyper + ischemia group were significantly higher than those in the Normo + ischemia group. HO-1 immunoreactivity in the Hyper + ischemia group was significantly increased in pyramidal neurons and astrocytes with time after ischemia, and the immunoreactivity was significantly higher than that in the Normo + ischemia group. In the Normo + Ischemia group, neuronal death was shown in pyramidal neurons located only in CA1 at 5 days after ischemia. However, in the Hyper + ischemia group, pyramidal neuronal death occurred in CA1-3 at 2 days after ischemia. Taken together, our findings showed that brain ischemic insult during hyperthermic condition brings up earlier and severer neuronal damage/death in the hippocampus, showing that HO-1 expression in neurons and astrocytes is different according to brain subregions and temperature condition. Based on these findings, we suggest that hyperthermia in patients with ischemic stroke must be taken into the consideration in the therapy.

CD200 Change Is Involved in Neuronal Death in Gerbil Hippocampal CA1 Field Following Transient Forebrain Ischemia and Postischemic Treatment with Risperidone Displays Neuroprotection without CD200 Change.

It has been reported that CD200 (Cluster of Differentiation 200), expressed in neurons, regulates microglial activation in the central nervous system, and a decrease in CD200 expression causes an increase in microglial activation and neuronal loss. The aim of this study was to investigate time-dependent changes in CD200 expression in the hippocampus proper (CA1, 2, and 3 fields) after transient forebrain ischemia for 5 min in gerbils. In this study, 5-min ischemia evoked neuronal death (loss) of pyramidal neurons in the CA1 field, but not in the CA2/3 fields, at 5 days postischemia. In the sham group, CD200 expression was found in pyramidal neurons of the CA1 field, and the immunoreactivity in the group with ischemia was decreased at 6 h postischemia, dramatically increased at 12 h postischemia, decreased (to level found at 6 h postischemia) at 1 and 2 days postischemia, and significantly increased again at 5 days postischemia. At 5 days postischemia, CD200 immunoreactivity was strongly expressed in microglia and GABAergic neurons. However, in the CA3 field, the change in CD200 immunoreactivity in pyramidal neurons was markedly weaker than that in the CA1 field, showing there was no expression of CD 200 in microglia and GABAergic neurons. In addition, treatment of 10 mg/kg risperidone (an atypical antipsychotic drug) after the ischemia hardly changed CD200 immunoreactivity in the CA1 field, showing that CA1 pyramidal neurons were protected from the ischemic injury. These results indicate that the transient ischemia-induced change in CD200 expression may be associated with specific and selective neuronal death in the hippocampal CA1 field following transient forebrain ischemia.

Increased Calbindin D28k Expression via Long-Term Alternate-Day Fasting Does Not Protect against Ischemia-Reperfusion Injury: A Focus on Delayed Neuronal Death, Gliosis and Immunoglobulin G Leakage.

Calbindin-D28k (CB), a calcium-binding protein, mediates diverse neuronal functions. In this study, adult gerbils were fed a normal diet (ND) or exposed to intermittent fasting (IF) for three months, and were randomly assigned to sham or ischemia operated groups. Ischemic injury was induced by transient forebrain ischemia for 5 min. Short-term memory was examined via passive avoidance test. CB expression was investigated in the Cornu Ammonis 1 (CA1) region of the hippocampus via western blot analysis and immunohistochemistry. Finally, histological analysis was used to assess neuroprotection and gliosis (microgliosis and astrogliosis) in the CA1 region. Short-term memory did not vary significantly between ischemic gerbils with IF and those exposed to ND. CB expression was increased significantly in the CA1 pyramidal neurons of ischemic gerbils with IF compared with that of gerbils fed ND. However, the CB expression was significantly decreased in ischemic gerbils with IF, similarly to that of ischemic gerbils exposed to ND. The CA1 pyramidal neurons were not protected from ischemic injury in both groups, and gliosis (astrogliosis and microgliosis) was gradually increased with time after ischemia. In addition, immunoglobulin G was leaked into the CA1 parenchyma from blood vessels and gradually increased with time after ischemic insult in both groups. Taken together, our study suggests that IF for three months increases CB expression in hippocampal CA1 pyramidal neurons; however, the CA1 pyramidal neurons are not protected from transient forebrain ischemia. This failure in neuroprotection may be attributed to disruption of the blood-brain barrier, which triggers gliosis after ischemic insults.

Therapeutic Effects of Decursin and Angelica gigas Nakai Root Extract in Gerbil Brain after Transient Ischemia via Protecting BBB Leakage and Astrocyte Endfeet Damage.

Angelica gigas Nakai root contains decursin which exerts beneficial properties such as anti-amnesic and anti-inflammatory activities. Until now, however, the neuroprotective effects of decursin against transient ischemic injury in the forebrain have been insufficiently investigated. Here, we revealed that post-treatment with decursin and the root extract saved pyramidal neurons in the hippocampus following transient ischemia for 5 min in gerbil forebrain. Through high-performance liquid chromatography, we defined that decursin was contained in the extract as 7.3 ± 0.2%. Based on this, we post-treated with 350 mg/kg of extract, which is the corresponding dosage of 25 mg/kg of decursin that exerted neuroprotection in gerbil hippocampus against the ischemia. In addition, behavioral tests were conducted to evaluate ischemia-induced dysfunctions via tests of spatial memory (by the 8-arm radial maze test) and learning memory (by the passive avoidance test), and post-treatment with the extract and decursin attenuated ischemia-induced memory impairments. Furthermore, we carried out histochemistry, immunohistochemistry, and double immunohistofluorescence. Pyramidal neurons located in the subfield cornu ammonis 1 (CA1) among the hippocampal subfields were dead at 5 days after the ischemia; however, treatment with the extract and decursin saved the pyramidal neurons after ischemia. Immunoglobulin G (IgG, an indicator of extravasation), which is not found in the parenchyma in normal brain tissue, was apparently shown in CA1 parenchyma from 2 days after the ischemia, but IgG leakage was dramatically attenuated in the CA1 parenchyma treated with the extract and decursin. Furthermore, astrocyte endfeet, which are a component of the blood-brain barrier (BBB), were severely damaged at 5 days after the ischemia; however, post-treatment with the extract and decursin dramatically attenuated the damage of the endfeet. In brief, therapeutic treatment of the extract of Angelica gigas Nakai root and decursin after 5 min transient forebrain ischemia protected hippocampal neurons from the ischemia, showing that ischemia-induced BBB leakage and damage of astrocyte endfeet was significantly attenuated by the extract and decursin. Based on these findings, we suggest that Angelica gigas Nakai root containing decursin can be employed as a pharmaceutical composition to develop a therapeutic strategy for brain ischemic injury.

Neuroprotective and Anti-Inflammatory Effects of Pinus densiflora Bark Extract in Gerbil Hippocampus Following Transient Forebrain Ischemia.

Korean red pine (Pinus densiflora) belongs to the Genus Pinus, and its bark contains a great amount of naturally occurring phenolic compounds. Until now, few studies have been conducted to assess the neuroprotective effects of Pinus densiflora bark extract against brain ischemic injury. The aim of this study was to investigate the neuroprotective effects of pre-treatment with the extract in the hippocampus following 5-min transient forebrain ischemia in gerbils. Furthermore, this study examined the anti-inflammatory effect as a neuroprotective mechanism of the extract. Pinus densiflora bark was extracted by pure water (100 °C), and this extract was quantitatively analyzed and contained abundant polyphenols, flavonoids, and proanthocyanidins. The extract (25, 50, and 100 mg/kg) was orally administered once a day for seven days before the ischemia. In the gerbil hippocampus, death of the pyramidal neurons was found in the subfield cornu ammonis 1 (CA1) five days after the ischemia. This death was significantly attenuated by pre-treatment with 100 mg/kg, not 25 or 50 mg/kg, of the extract. The treatment with 100 mg/kg of the extract markedly inhibited the activation of microglia (microgliosis) and significantly decreased the expression of pro-inflammatory cytokines (interleukin 1ß and tumor necrosis factor α). In addition, the treatment significantly increased anti-inflammatory cytokines (interleukin 4 and interleukin 13). Taken together, this study clearly indicates that pre-treatment with 100 mg/kg of Pinus densiflora bark extract in gerbils can exert neuroprotection against brain ischemic injury by the attenuation of neuroinflammatory responses.

Laminarin Attenuates Ultraviolet-Induced Skin Damage by Reducing Superoxide Anion Levels and Increasing Endogenous Antioxidants in the Dorsal Skin of Mice.

A number of studies have demonstrated that marine carbohydrates display anti-oxidant, anti-melanogenic, and anti-aging activities in the skin. Laminarin (LA), a low-molecular-weight polysaccharide, is found in brown algae. The benefits of LA in ultraviolet B (UVB) induced photodamage of the skin have not been reported. The aim of this study was to investigate the effects of pre-treated LA on histopathological changes and oxidative damage in mouse dorsal skin on day 5, following repeated UVB exposure. Histopathology, Western blot analysis and immunohistochemical studies showed that epidermal thickness in the UVB group was significantly increased; however, the thickness in the UVB group treated with LA (LA/UVB group) was less compared with that of the UVB group. Collagen fibers in the dermis of the UVB group were significantly decreased and destroyed, whereas, in the LA/UVB group, the density of collagen fibers was significantly increased compared with that of the UVB group. Oxidative stress due to superoxide anion production measured via dihydroethidium fluorescence staining was dramatically increased in the UVB group, whereas in the LA/UVB group, the oxidative stress was significantly decreased. Expressions of SOD1, glutathione peroxidase and catalase were markedly reduced in the UVB group, whereas in the LA/UVB group, they were significantly higher along with SOD2 than in the control group. Taken together, our results indicate that LA pretreatment prevents or attenuates skin damage, by decreasing oxidative stress and increasing antioxidant enzymes in mouse dorsal skin.

Effects of Decursin and Angelica gigas Nakai Root Extract on Hair Growth in Mouse Dorsal Skin via Regulating Inflammatory Cytokines.

This current study investigates the facilitative effects and mechanisms of decursin, a major component of Angelica gigas Nakai (AGN), and AGN root extract on hair growth in mice. We perform high-performance liquid chromatography on AGN extract to show it contains 7.3% decursin. Hairs in mouse dorsal skin are shaved distilled in water, 0.15% decursin, and 2% AGN root extract (0.15% decursin in the diluted extract) and topically applied twice a day for 17 days. Hematoxylin and eosin staining are done to examine the morphological changes in the hair follicles. To compare the effects of decursin and AGN extract on inflammatory cytokines in the dorsal skin, Western blot analysis and immunohistochemistry for tumor necrosis factor α (TNF-α) and interleukin (IL)-1ß as pro-inflammatory cytokines, and IL-4 and IL-13 as anti-inflammatory cytokines are conducted. The results show that the application of decursin and AGN extract confer effects on hair growth. Hair growth is significantly facilitated from seven days after the treatments compared to that in the control group, and completely grown hair was found 17 days after the treatments. The protein levels and immunoreactivity of TNF-α and IL-1ß in this case are significantly decreased, whereas the IL-4 and IL-13 levels and immunoreactivity are significantly increased compared to those in the control group. Additionally, high-mobility group box 1, an inflammatory mediator, is elevated by the topical application of decursin and AGN extract. Taken together, the treatment of mouse dorsal skin with AGE root extract containing decursin promotes hair growth by regulating pro- and/or anti-inflammatory cytokines. We, therefore, suggest that AGN root extract as well as decursin can be utilized as materials for developing hair growth-facilitating treatments.

Chemical Composition of a Novel Distillate from Fermented Mixture of Nine Anti-Inflammatory Herbs and Its UVB-Protective Efficacy in Mouse Dorsal Skin via Attenuating Collagen Disruption and Inflammation.

Since ancient times, various herbs have been used in Asia, including Korea, China, and Japan, for wound healing and antiaging of the skin. In this study, we manufactured and chemically analyzed a novel distillate obtained from a fermented mixture of nine anti-inflammatory herbs (Angelica gigas, Lonicera japonica, Dictamnus dasycarpus Turcz., D. opposita Thunb., Ulmus davidiana var. japonica, Hordeum vulgare var. hexastichon Aschers., Xanthium strumarium L., Cnidium officinale, and Houttuynia cordata Thunb.). The fermentation of natural plants possesses beneficial effects in living systems. These activities are attributed to the chemical conversion of the parent plants to functional constituents which show more potent biological activities. In our current study, the distillate has been manufactured after fermenting the nine oriental medical plants with Lactobacillus fermentum, followed by distilling. We analyzed the chemical ingredients involved in the distillate and evaluated the effects of topical application of the distillate on ultraviolet B (UVB)-induced skin damage in Institute of Cancer Research (ICR) mice. Topical application of the distillate significantly ameliorated the macroscopic and microscopic morphology of the dorsal skin against photodamage induced by UVB radiation. Additionally, our current results showed that topical application of the distillate alleviated collagen disruption and reduced levels of proinflammatory cytokines (tumor necrosis factor alpha and interleukin 1 ß expressions) in the dorsal skin against UVB radiation. Taken together, our current findings suggest that the distillate has a potential to be used as a material to develop a photoprotective adjuvant.

Topical Application of Aronia melanocarpa Extract Rich in Chlorogenic Acid and Rutin Reduces UVB-Induced Skin Damage via Attenuating Collagen Disruption in Mice.

Aronia melanocarpa, a black chokeberry, contains high levels of phenolic acids and polyphenolic flavonoids and displays antioxidative and anti-inflammatory effects. Through high-performance liquid chromatography for extracts from Aronia melanocarpa, we discovered that the extract contained chlorogenic acid and rutin as major ingredients. In this study, we examined the protective effects of the extract against ultraviolet B- (UVB)-induced photodamage in the dorsal skin of institute of cancer research (ICR) mice. Their dorsal skin was exposed to UVB, thereafter; the extract was topically applied once a day for seven days. Photoprotective properties of the extract in the dorsal skin were investigated by clinical skin severity score for skin injury, hematoxylin and eosin staining for histopathology, Masson's trichrome staining for collagens. In addition, we examined change in collagen type I and III, and matrix metalloproteinase (MMP)-1 and MMP-3 by immunohistochemistry. In the UVB-exposed mice treated with the extract, UVB-induced epidermal damage was significantly ameliorated, showing that epidermal thickness was moderated. In these mice, immunoreactivities of collagen type I and III were significantly increased, whereas immunoreactivities of MMP-1 and 3 were significantly decreased compared with those in the UVB-exposed mice. These results indicate that treatment with Aronia melanocarpa extract attenuates UV-induced photodamage by attenuating UVB-induced collagen disruption: these findings might be a result of the chlorogenic acid and rutin contained in the extract. Based on the current results, we suggest that Aronia melanocarpa can be a useful material for developing photoprotective adjuvant.

Validation of the Korean Version of the Community Assessment of Psychic Experiences in General Population.

OBJECTIVE: The Community Assessment of Psychic Experiences has been widely translated and commonly used as a measure for psychotic experiences and psychosis proneness in clinical and research environments worldwide. This study aimed to establish the psychometric properties (reliability and validity) and factor structure of a Korean version of the Community Assessment of Psychic Experiences (K-CAPE) in the general population. METHODS: A total of 1,467 healthy participants completed K-CAPE and other psychiatric symptom-related scales (Paranoia scale, Patient Health Questionnaire-9, Dissociative Experiences Scale-II, and Oxford-Liverpool Inventory of Feelings and Experiences) via online survey. K-CAPE's internal reliability was analyzed using Cronbach's alpha coefficient. Confirmatory factor analysis (CFA) was performed to investigate whether the original three-factor model (positive, negative, and depressive) and other hypothesized multidimensional models (including positive and negative subfactors) were suitable for our data. Exploratory factor analysis (EFA) was conducted to explore better alternative factor solutions with a follow-up CFA. To assess convergent and discriminant validity, we examined correlations between K-CAPE subscales with other established measures of psychiatric symptoms. RESULTS: K-CAPE showed good internal consistency in all original three subscales (all greater than α=0.827). The CFA demonstrated that the multidimensional models exhibited relatively better quality than the original three-dimensional model. Although the model fit indices did not reach their respective optimal thresholds, they were within an acceptable range. Results from the EFA indicated 3-5 factor solutions. In 3-factor solution, "negative-avolition" items were founded to be loaded more consistently with depressive items than with the negative dimension. In 4-factor solution, positive items were divided into two subfactors: "positive-bizarre experiences" and "positive-delusional thoughts," while negative symptoms were separated into two distinct subfactors in 5-factor solution: "negative-avolition (expressive)," and "negative-social (experiential)." The correlation coefficients between K-CAPE subscales and corresponding measurements were significant (p<0.001), confirming the convergent and discriminant validity. CONCLUSION: Our study provides evidence to support the reliability and validity of the K-CAPE and its use as a measure of psychotic symptoms in the Korean population. Although alternative factor structures did not improve the model fit, our EFA findings implicate the use of subfactors to investigate more specific domains of positive and negative symptoms. Given the heterogeneous nature of psychotic symptoms, this may be useful in capturing their different underlying mechanisms.

Associations between post-traumatic stress disorders and psychotic symptom severity in adult survivors of developmental trauma: a multisite cross-sectional study in the UK and South Korea.

BACKGROUND: Childhood maltreatment is a risk factor for the development of post-traumatic stress disorders and psychosis. However, the association between post-traumatic stress disorder (PTSD), including complex PTSD, and psychotic symptoms is unknown. We investigated whether the presence of PTSD and complex PTSD was associated with psychotic symptom severity within survivors of developmental trauma. METHODS: As part of the Investigating Mechanisms underlying Psychosis Associated with Childhood Trauma (IMPACT) study, from Aug 20, 2020, to Jan 24, 2021, and from Sept 9, 2022, to Feb 21, 2023, using study advertisement on online platforms we recruited adult (≥18 years) participants who had experienced developmental trauma without a psychiatric diagnosis in the UK and South Korea. We measured whether participants met diagnostic thresholds for PTSD and complex PTSD using the self-reported International Trauma Questionnaire, and psychotic symptoms using the self-reported Community Assessment of Psychic Experiences. We used linear regression, adjusting for sociodemographic variables such as age, sex, ethnicity, educational attainment, and socioeconomic status, to examine whether there was an association between PTSD and complex PTSD and psychotic symptoms. The study is registered in the UK (University College London Research Ethics Committee [14317/001] and the National Health Service Research Ethics Committee [22/YH/0096]) and South Korea (Institutional Review Board of Seoul National University Bundang Hospital [B-2011-648-306]), and is ongoing. FINDINGS: Of the 2675 participants who took part in the study, 1273 had experienced developmental trauma and were included in the study in the UK (n=475) and South Korea (n=798), comprising 422 (33%) men and 851 (67%) women with a mean age of 26·9 years (SD 6, range 18-40), mostly of White British (n=328) or South Korean (n=798) ethnicity. We found no significant association between PTSD and psychotic symptom severity (total severity ß=-2·40 [SE 3·28], p=0·47), compared with participants who did not meet PTSD or complex PTSD caseness. We found a significant relationship between complex PTSD and psychotic symptom severity (total severity ß=22·62 [SE 1·65], p<0·0001), including for positive (ß=12·07 [SE 0·99], p<0·0001) and negative symptoms (ß=10·5 [SE 0·95], p<0·0001), compared with participants who did not meet PTSD or complex PTSD caseness. INTERPRETATION: Health systems must assess individuals with previous developmental trauma for complex PTSD and treat those affected. These individuals should also be assessed for psychotic symptoms, and if necessary, preventative measures should be taken to reduce risk of conversion. Further work should assess whether treating complex PTSD modifies the risk of conversion to psychosis. FUNDING: UKRI Future Leaders Fellowship, British Medical Association Margaret Temple Award for Schizophrenia Research, and the National Research Foundation of Korea-Korea Government.

Hyperthermia accelerates neuronal loss differently between the hippocampal CA1 and CA2/3 through different HIF­1α expression after transient ischemia in gerbils.

The hippocampus has a different vulnerability to ischemia according to the subfields CA1 to CA3 (initials of cornu ammonis). It has been reported that body temperature changes during ischemia affect the degree of neuronal death following transient ischemia. Hypoxia­inducible factor 1α (HIF­1α) plays a key role in regulating cellular adaptation to low oxygen conditions. In the present study, we investigated the pattern of neuronal death (loss) in CA1 and CA2/3 following 5 min transient forebrain ischemia (TFI) under hyperthermia (39.5±0.2˚C) and the relationship between neuronal death and changes in HIF­1α expression using western blot analysis and immunohistochemistry in gerbils. Normothermia or hyperthermia was induced for 30 min before and during the TFI, and neuronal death and HIF­1α expression were observed at 0, 3, 6 and 12 h, 1, 2 and 5 days after TFI. Under normothermia, TFI­induced neuronal death of CA1 pyramidal neurons occurred on day 5 after TFI, but CA2/3 pyramidal neurons did not die. In contrast, under hyperthermia, the death of CA1 and CA2/3 pyramidal neurons was observed on day 2 after TFI. Under normothermia, HIF­1α expression was significantly elevated in both CA1 and CA2/3 pyramidal neurons at 12 h and 1 day after TFI, and the increased HIF­1α immunoreactivity in CA1 was dramatically reduced from 2 days after TFI, but not in CA2/3 pyramidal neurons. Under hyperthermia, the basal expression of HIF­1α in the sham group was significantly higher in both CA1 and CA2/3 pyramidal neurons at 0 h after TFI than in the normothermia group. HIF­1 expression was continuously higher, peaked at 12 h after TFI, and then significantly decreased from 1 day after TFI. Overall, the present results indicate that resistance to ischemia in CA2/3 pyramidal neurons is closely associated with the persistence of increased expression of HIF­1α after ischemic insults and that hyperthermia­induced exacerbation of death of pyramidal neurons is closely related to decreased HIF­1α expression after ischemic insults.

Pinus thunbergii bark extract rich in flavonoids promotes hair growth in dorsal skin by regulating inflammatory cytokines and increasing growth factors in mice.

Korean maritime pine bark (Pinus thunbergii) has been used as an alternative medicine due to its beneficial properties, including anti­inflammatory effects. To date, the anti­inflammatory and hair growth­promoting effects of Pinus densiflora bark extract have remained elusive. Therefore, in the present study, Pinus thunbergii bark was extracted with pure water (100˚C) and the extract was examined to determine its polyphenol and flavonoid content. C57BL/6 mice were used to assess the effects of the extract to promote hair growth. The extract (1, 2 and 4%) was topically applied onto shaved dorsal skin and hair growth was observed for 17 days. A significant increase in hair growth was observed with 2 and 4% extract. Based on this finding, the optimal dose of the extract for effective hair growth promotion was determined to be 2%. The mechanisms of hair growth promotion were investigated via immunohistochemical analysis of changes in inflammatory cytokines and growth factors in the hair follicles following treatment with 2% extract. The treatment reduced the levels of TNF­α and IL­1ß, which are pro­inflammatory cytokines, while it enhanced the levels of IL­4 and IL­13, which are anti­inflammatory cytokines, in the hair follicles. In addition, elevated insulin­like growth factor I and vascular epidermal growth factor were detected in hair follicles following treatment. Based on these findings, it was suggested that the extract of Pinus thunbergii bark may be utilized for hair loss prevention and/or hair growth promotion.

Hypothermic treatment reduces matrix metalloproteinase-9 expression and damage in the liver following asphyxial cardiac arrest in rats.

BACKGROUND: Hypothermic treatment is known to protect organs against cardiac arrest (CA) and improves survival rate. However, few studies have evaluated the effects of hypothermia on CA-induced liver damages. This study was designed to analyzed the possible protective effects of hypothermia on the liver after asphyxial CA (ACA). Rats were randomly subjected to 5 min of ACA followed by return of spontaneous circulation (ROSC). Body temperature was controlled at 37 ± 0.5 °C (normothermia group) or 33 ± 0.5 °C (hypothermia group) for 4 h after ROSC. Liver tissues were extracted and examined at 6 h, 12 h, 1 day, and 2 days after ROSC. RESULTS: The expression of infiltrated neutrophil marker CD11b and matrix metallopeptidase-9 (MMP9) was investigated via immunohistochemistry. Morphological damage was assessed via hematoxylin and eosin (H & E) staining. Hypothermic treatment improved the survival rate at 6 h, 12 h, 1 day, and 2 days after ACA. Based on immunohistochemical analysis, the expression of CD11b and MMP9 was significantly increased from 6 h after ACA in the normothermia group. However, the expressions of CD11b and MMP9 was significantly decreased in the hypothermia group compared with that of the normothermia group. In addition, in the results of H & E, sinusoidal dilatation and vacuolization were apparent after ACA; however, these ACA-induced structural changes were reduced by the 4 h-long hypothermia. CONCLUSIONS: In conclusion, hypothermic treatment for 4 h inhibited the increases in CD11b and MMP9 expression and reduced the morphological damages in the liver following ACA in rats. This study suggests that hypothermic treatment after ACA reduces liver damages by regulating the expression of CD11b and MMP9.

Differences in TNF­α and TNF­R1 expression in damaged neurons and activated astrocytes of the hippocampal CA1 region between young and adult gerbils following transient forebrain ischemia.

Tumor necrosis factor (TNF)­α and TNF receptor 1 (TNF­R1) play diverse roles in modulating the neuronal damage induced by cerebral ischemia. The present study compared the time­dependent changes of TNF­α and TNF­R1 protein expression levels in the hippocampal subfield cornu ammonis 1 (CA1) between adult and young gerbils following transient forebrain ischemia (tFI), via western blot and immunohistochemistry analyses. In adult gerbils, delayed neuronal death of pyramidal neurons, the principal neurons in CA1, was recorded 4 days after tFI; however, in young gerbils, delayed neuronal death was recorded 7 days after tFI. TNF­α protein expression levels gradually increased in both groups following tFI; however, TNF­α expression was higher in young gerbils compared with adult gerbils. TNF­R1 protein expression levels markedly increased in both groups 1 day after tFI. Subsequently, TNF­R1 expression gradually decreased in young gerbils, whereas TNF­R1 expression levels were irregularly altered in adult gerbils following tFI. Notably, TNF­α immunoreactivity significantly increased in pyramidal neurons in both groups 1 day after tFI; however, the patterns altered between both groups. In adult gerbils, TNF­α immunoreactivity was rarely exhibited in pyramidal neurons 4 days after tFI due to neuronal death, suggesting that TNF­α immunoreactivity was newly expressed in astrocytes. In young gerbils, TNF­α immunoreactivity increased in pyramidal neurons 4 days after tFI, and TNF­α immunoreactivity was newly expressed in astrocytes. In addition, TNF­R1 immunoreactivity was exhibited in pyramidal cells of both sham groups, and significantly increased 1 day after tFI; however, the patterns altered between both groups. In adult gerbils, TNF­R1 immunoreactivity was rarely exhibited 4 days after tFI, and astrocytes newly expressed TNF­R1 immunoreactivity. In young gerbils, TNF­R1 immunoreactivity increased in pyramidal neurons 4 days after tFI; however, TNF­R1 immunoreactivity was not reported in pyramidal neurons and astrocytes thereafter. Taken together, the results of the present study suggest that different expression levels of TNF­α and TNF­R1 in ischemic CA1 between adult and young gerbils may be due to age­dependent differences of tFI­induced neuronal death.

Ischemia-Induced Cognitive Impairment Is Improved via Remyelination and Restoration of Synaptic Density in the Hippocampus after Treatment with COG-Up® in a Gerbil Model of Ischemic Stroke.

Cerebrovascular disease such as ischemic stroke develops cognitive impairment due to brain tissue damage including neural loss, demyelination and decrease in synaptic density. In the present study, we developed transient ischemia in the forebrain of the gerbil and found cognitive impairment using the Barnes maze test and passive avoidance test for spatial memory and learning memory, respectively. In addition, neuronal loss/death was detected in the Cornu Ammonis 1 (CA1) region of the gerbil hippocampus after the ischemia by cresyl violet histochemistry, immunohistochemistry for neuronal nuclei and histofluorescence with Fluoro-Jade B. Furthermore, in the CA1 region following ischemia, myelin and vesicular synaptic density were significantly decreased using immunohistochemistry for myelin basic protein and vesicular glutamate transporter 1. In the gerbils, treatment with COG-up® (a combined extract of Erigeron annuus (L.) Pers. and Brassica oleracea Var.), which was rich in scutellarin and sinapic acid, after the ischemia, significantly improved ischemia-induced decline in memory function when compared with that shown in gerbils treated with vehicle after the ischemia. In the CA1 region of these gerbils, COG-up® treatment significantly promoted the remyelination visualized using immunohistochemistry myelin basic protein, increased oligodendrocytes visualized using a receptor-interacting protein, and restored the density of glutamatergic synapses visualized using double immunofluorescence for vesicular glutamate transporter 1 and microtubule-associated protein, although COG-up® treatment did not protect pyramidal cells (principal neurons) located in the CA1 region form the ischemic insult. Considering the current findings, a gerbil model of ischemic stroke apparently showed cognitive impairment accompanied by ischemic injury in the hippocampus; also, COG-up® can be employed for improving cognitive decline following ischemia-reperfusion injury in brains.

Neuroprotective Effects of Salicin in a Gerbil Model of Transient Forebrain Ischemia by Attenuating Oxidative Stress and Activating PI3K/Akt/GSK3ß Pathway.

Salicin is a major natural compound of willow bark and displays diverse beneficial biological properties, such as antioxidant activity. However, little information available for the neuroprotective potential of salicin against ischemic brain injury has been reported. Thus, this study was performed to investigate the neuroprotective potential of salicin against ischemia and reperfusion (IR) injury and its mechanisms in the hippocampus using a gerbil model of 5-min transient ischemia (TI) in the forebrain, in which a massive loss (death) of pyramidal neurons cells occurred in the subfield Cornu Ammonis 1 (CA1) among the hippocampal subregions (CA1-3) at 5 days after TI. To examine neuroprotection by salicin, gerbils were pretreated with salicin alone or together with LY294002, which is a phosphatidylinositol 3-kinase (PI3K) inhibitor, once daily for 3 days before TI. Treatment with 20 mg/kg of salicin significantly protected CA1 pyramidal neurons against the ischemic injury. Treatment with 20 mg/kg of salicin significantly reduced the TI-induced increase in superoxide anion generation and lipid peroxidation in the CA1 pyramidal neurons after TI. The treatment also reinstated the TI-induced decrease in superoxide dismutases (SOD1 and SOD2), catalase, and glutathione peroxidase in the CA1 pyramidal cells after TI. Moreover, salicin treatment significantly elevated the levels of phosphorylation of Akt and glycogen synthase kinase-3ß (GSK3ß), which is a major downstream target of PI3K, in the ischemic CA1. Notably, the neuroprotective effect of salicin was abolished by LY294002. Taken together, these findings clearly indicate that salicin protects against ischemic brain injury by attenuating oxidative stress and activating the PI3K/Akt/GSK3ß pathway.

Comparison of age-dependent alterations in thioredoxin 2 and thioredoxin reductase 2 expressions in hippocampi between mice and rats.

BACKGROUND: Aging is one of major causes triggering neurophysiological changes in many brain substructures, including the hippocampus, which has a major role in learning and memory. Thioredoxin (Trx) is a class of small redox proteins. Among the Trx family, Trx2 plays an important role in the regulation of mitochondrial membrane potential and is controlled by TrxR2. Hitherto, age-dependent alterations in Trx2 and TrxR2 in aged hippocampi have been poorly investigated. Therefore, the aim of this study was to examine changes in Trx2 and TrxR2 in mouse and rat hippocampi by age and to compare their differences between mice and rats. RESULTS: Trx2 and TrxR2 levels using Western blots in mice were the highest at young age and gradually reduced with time, showing that no significant differences in the levels were found between the two subfields. In rats, however, their expression levels were the lowest at young age and gradually increased with time. Nevertheless, there were no differences in cellular distribution and morphology in their hippocampi when it was observed by cresyl violet staining. In addition, both Trx2 and TrxR2 immunoreactivities in the CA1-3 fields were mainly shown in pyramidal cells (principal cells), showing that their immunoreactivities were altered like changes in their protein levels. CONCLUSIONS: Our current findings suggest that Trx2 and TrxR2 expressions in the brain may be different according to brain regions, age and species. Therefore, further studies are needed to examine the reasons of the differences of Trx2 and TrxR2 expressions in the hippocampus between mice and rats.

Neuronal Death in the CNS Autonomic Control Center Comes Very Early after Cardiac Arrest and Is Not Significantly Attenuated by Prompt Hypothermic Treatment in Rats.

Autonomic dysfunction in the central nervous system (CNS) can cause death after recovery from a cardiac arrest (CA). However, few studies on histopathological changes in animal models of CA have been reported. In this study, we investigated the prevalence of neuronal death and damage in various brain regions and the spinal cord at early times after asphyxial CA and we studied the relationship between the mortality rate and neuronal damage following hypothermic treatment after CA. Rats were subjected to 7-8 min of asphyxial CA, followed by resuscitation and prompt hypothermic treatment. Eight regions related to autonomic control (the cingulate cortex, hippocampus, thalamus, hypothalamus, myelencephalon, and spinal cord) were examined using cresyl violet (a marker for Nissl substance) and Fluoro-Jade B (a marker for neuronal death). The survival rate was 44.5% 1 day post-CA, 18.2% 2 days post-CA and 0% 5 days post-CA. Neuronal death started 12 h post-CA in the gigantocellular reticular nucleus and caudoventrolateral reticular nucleus in the myelencephalon and lamina VII in the cervical, thoracic, lumbar, and sacral spinal cord, of which neurons are related to autonomic lower motor neurons. In these regions, Iba-1 immunoreactivity indicating microglial activation (microgliosis) was gradually increased with time after CA. Prompt hypothermic treatment increased the survival rate at 5 days after CA with an attenuation of neuronal damages and death in the damaged regions. However, the survival rate was 0% at 12 days after CA. Taken together, our study suggests that the early damage and death of neurons related to autonomic lower motor neurons was significantly related to the high mortality rate after CA and that prompt hypothermic therapy could increase the survival rate temporarily after CA, but could not ultimately save the animal.