Shunichiro Okazaki

Femoral head osteonecrosis can be caused by disruption of the systemic immune response via the toll-like receptor 4 signalling pathway

OBJECTIVES Osteonecrosis of the femoral head is observed in patients treated with steroids. However, the pathogenesis of femoral head osteonecrosis remains unclear. We established a rat model with femoral head osteonecrosis by injecting lipopolysaccharide (LPS) and steroid, and assessed the consequences of this on femoral head histology, the systemic immune response and lipid synthesis. METHODS Male Wistar rats were injected intravenously on days 0 and 1 with 2 mg/kg LPS and intramuscularly with 20 mg/kg methylprednisolone on days 3, 4 and 5. The animals were sacrificed 1, 2, 3 or 4 weeks after the last methylprednisolone injection. Histopathological and biochemical analyses were performed every week. RESULTS Osteonecrosis of the femoral head was observed in the rats. The plasma triglyceride concentrations had decreased significantly by weeks 2 and 3. The total plasma cholesterol concentrations had increased significantly by week 1 but then decreased significantly by week 4. The plasma concentrations of IL-1beta, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-gamma and TNF-alpha had increased significantly by week 1. These cytokines can all be induced by toll-like receptor 4 (TLR4) signalling. CONCLUSIONS LPS and methylprednisolone induced osteonecrosis of the femoral head in rats and this was associated with a disruption of the innate immune system and lipid synthesis. These findings suggest that the TLR4 signalling pathway plays an important role in the pathogenesis of femoral head osteonecrosis.

Prior ethanol injection promotes brain edema after traumatic brain injury

Alcohol consumption prior to traumatic brain injury (TBI) promotes morbidity and mortality although the mechanisms involved remain unclear. The morbidity and mortality caused by TBI, especially brain contusion, are known to be closely associated with brain edema. Here we examined the effects of ethanol pretreatment on brain edema, inflammatory responses, and oxidative stress after brain contusion. Male Wistar rats were given 3 g/kg ethanol intraperitoneally and 1 h later were subjected to brain contusion. The ethanol-pretreated group had a significantly decreased survival rate. Magnetic resonance imaging showed ethanol pretreatment significantly augmented the volume of cytotoxic brain edema after contusion. In the ethanol-pretreated rat, the activities of NF-kappaB and AP-1 were reduced 6 h after contusion and COX-2 mRNA expression was increased 24 h after contusion. These findings suggest that ethanol augmented cerebral edema and mortality in rats with brain contusion, possibly through actions on cell survival pathways or COX-2 expression. In addition, antioxidant treatment at 3 h post-injury significantly attenuated some markers of oxidative stress, mortality, and volume of edema at 24 h after ethanol treatment and contusion.

Experimental rat model for alcohol‐induced osteonecrosis of the femoral head

Alcohol‐induced osteonecrosis of the femoral head (ONFH) is observed in alcohol abusers and patients with alcoholic fatty liver disease. It has been reported that Toll‐like receptor 4 (TLR4) signalling plays a crucial role in the pathogenesis of alcoholic fatty liver disease. We previously reported a corticosteroid‐induced ONFH rat model, and suggested that TLR4 signalling contributes to the pathogenesis of ONFH. Thus, it is thought that the pathogenesis of alcohol‐induced ONFH is probably similar to that of corticosteroid‐induced ONFH. The aim of this study was to develop a new animal model for alcohol‐induced ONFH and to evaluate the relationship between the pro‐inflammatory response via TLRs and the development of ONFH in rats. Male Wistar rats were fed a Lieber–DeCarli liquid diet containing 5% ethanol (experimental group) or dextran (control group) for 1–24 weeks. Histopathological and biochemical analyses were performed. Feeding the ethanol‐containing liquid diet resulted in the development of ONFH with hepatic steatosis, hepatic dysfunction and hyperlipidaemia, whereas feeding the dextran‐containing diet did not cause ONFH. However, we could not recognize any relationship between the pro‐inflammatory response via TLR4 and the development of alcohol‐induced ONFH. Thus in this study we have developed a new rat model for alcohol‐induced ONFH based on the feeding of an ethanol liquid diet. ONFH was observed within seven days from the start of feeding with 5% ethanol‐containing liquid diet. Although this was linked to hepatic steatosis, a TLR4 association was not a feature of this model.

Time-related course of pleural space fluid collection and pulmonary aeration on postmortem computed tomography (PMCT)

Postmortem CT (PMCT) is increasingly used in forensic practice, and knowledge and classification of typical postmortem imaging findings would facilitate the interpretation of PMCT. The goal of this study was to define the time-related course of postmortem chest findings. Twelve cadavers (eight male, four female, 27-81 [mean, 60.0]years) were examined twice by PMCT within an interval of time (4-164 h [mean, 30.8; median, 17.5]). The pleural-space-fluid volume, pulmonary parenchyma volume, decreased aerated lung volume (DLV), %DLV (=DLV/pulmonary parenchyma volume) and chest cavity volume were compared between the first and second PMCT examinations. To evaluate the volume change rate, the rate of increase in pleural space fluid volume (mL/h) and the DLV rate (mL/h) were plotted according to the postmortem period. On the second PMCT, the volume of pleural space fluid (p=0.0469) and %DLV (p=0.0161) were significantly increased. The increase rate of the pleural space fluid increased at approximately 30 h and the volume continued to increase until approximately 40 h after death. The rate of DLV constantly decreased in the early postmortem period. In conclusion, the pleural-space-fluid collection and the DLV increased over different time-related courses in the postmortem period.

New pitfalls of high-density postmortem computed tomography

An 80-year-old female was transferred to the hospital due to a traffic accident. Multiple cranial bone fractures with intracranial hemorrhage and intracranial air were detected. Despite treatment, the patient died after 6h. Twenty-one hours after the patient died, her whole body was scanned by postmortem CT, and a region of high density was detected within the left putamen. The autopsy revealed a cerebral contusion and multiple skull base fractures. Moreover, superabsorbent polymers (SAPs) were found within the left lateral ventricle and adjacent to the putamen, which appeared as a high-density lesion on postmortem CT at the left putamen, where the SAPs were compacted. Both ante- and postmortem conditions should be considered to prevent misdiagnoses based only on postmortem CT.

Acid-sensing ion channel 3 or P2X2/3 is involved in the pain-like behavior under a high bone turnover state in ovariectomized mice.

We have recently demonstrated that pathological changes leading to increased bone resorption by osteoclast activation are related to the induction of pain‐like behavior in ovariectomized (OVX) mice. In addition, bisphosphonate and the antagonist of transient receptor potential vanilloid type 1 (TRPV1), an acid‐sensing nociceptor, improved the threshold value of pain‐like behaviors accompanying an improvement in the acidic environment in the bone tissue based on osteoclast inactivation. The aim of this study was to evaluate the effect of (i) an inhibitor of vacuolar H+‐ATPase, known as an proton pump, (ii) an antagonist of acid‐sensing ion channel (ASIC) 3, as another acid‐sensing nociceptor, and (iii) the P2X2/3 receptor, as an ATP‐ligand nociceptor, on pain‐like behavior in OVX mice. This inhibitor and antagonists were found to improve the threshold value of pain‐like behavior in OVX mice. These results indicated that the skeletal pain accompanying osteoporosis is possibly associated with the acidic microenvironment and increased ATP level caused by osteoclast activation under a high bone turnover state.

The suppression of TRIM21 and the accumulation of IFN- α play crucial roles in the pathogenesis of osteonecrosis of the femoral head

Osteonecrosis of the femoral head (ONFH), the pathogenesis of which remains unclear, has been observed in autoimmune disease patients treated with corticosteroids. Recently, it has been shown that anti-tripartite motif-containing 21 (TRIM21) autoantibodies, which are often present in patients with systemic lupus erythematosis and Sjögrens syndrome, inhibit the E3 ligase activity of TRIM21. TRIM21 negatively regulates nuclear factor-κB (NF-κB) and interferon regulatory factors (IRFs) 3 and 7, three downstream transcription factors, via toll-like receptor 4 signaling. The aim of this study was to clarify the role of TRIM21 in the pathogenesis of ONFH using an animal model. Male Wistar rats were injected with lipopolysaccharide (LPS) twice and with methylprednisolone (MPSL) or saline three times. N-acetyl cysteine (NAC) was administered either concurrently with MPSL or once daily for the 3 days following the last MPSL injection. The incidence of ONFH in the MPSL group was 23.5%. Co-treatment of NAC and MPSL increased the incidence of ONFH to 55.6%. MPSL treatment decreased the activity of NF-κB in the liver and significantly increased the activity of both IRF3 and IRF7. No significant differences were observed in the activity of any of these three transcription factors between the MPSL and the co-treatment groups. In the femoral head, co-treatment with NAC and MPSL significantly decreased the expression of TRIM21 at 3 h and significantly increased the expression of interferon (IFN)-α at 24 h when compared with the MPSL group. IFN-α is known to induce cell death. These findings suggest that the suppression of TRIM21 in the femoral head causes an accumulation of IFN-α, which in turn leads to the development of ONFH. In conclusion, the suppression of TRIM21 resulting from altered NF-κB and IRF homeostasis accelerates the ONFH in rats treated with corticosteroids following LPS administration.

Weight bearing does not contribute to the development of osteonecrosis of the femoral head

The hip joint is one of the major structures in the human body and the resultant force acting through the hip joint is 300% of body weight. Therefore, weight bearing, as a cause of ischaemia, may contribute to the development of non‐traumatic osteonecrosis of the femoral head (ONFH). However, it remains unclear whether weight bearing is related to the development of non‐traumatic ONFH. Therefore the aim of this study was to clarify the role of weight bearing in the development of non‐traumatic ONFH. Non‐weight‐bearing (NWB) rats were tail suspended to prevent any weight coming to bear on the hindlimbs from day 1 to the time of sacrifice. The weight‐bearing (WB) group rats were also housed individually, although without tail suspension. All rats were injected with lipopolysaccharide and methylprednisolone to promote the development of non‐traumatic ONFH. All animals were sacrificed three weeks after the final methylprednisolone injection. Histopathological analysis was performed. Osteonecrosis of the femoral head was observed not only in the NWB but also in the WB rats; however, no osteonecrosis of the humeral head was observed in either group. We confirmed that non‐traumatic ONFH developed in NWB rats, suggesting that weight bearing does not contribute to the development of non‐traumatic ONFH in rats.

Postmortem computed tomography findings in the thorax – Experimental evaluation

OBJECTIVE Experimental fatal models were prepared to investigate the time-related course of lung changes using postmortem CT (PMCT). This study was approved by our institutional animal ethics committee. MATERIALS AND METHODS Twenty-four NZW rabbits (female 24, 2.30-4.30 (mean 3.10)kg) were divided into 4 fatal groups; drowning, hypothermia, bag suffocation, and Potassium Chloride intravenous (control) group. All individuals were examined by CT (Aquilion CX, Toshiba, Japan) on postmortem time course until detection of putrefaction air. The percent of aerated lung volume (%ALV=100*(ALV/total lung volume)) was measured and the pleural space fluid was investigated by axial imaging. A paired t-test and Bonferroni/Dunn study were employed for statistical evaluation. RESULTS In intra-group analysis, the %ALV showed statistically different periods compared with each pre-image: 4-48 h in control, 1-24h in drowning, 5-6h in hypothermia, and 1-4h in bag suffocation. In inter-group comparison (compared with control group), the %ALV increased in suffocation and decreased in drowning within 12h. The %ALV remained significantly high in hypothermia until 24h. The earliest detection times of pleural space fluid collection were different in each group: control (20 h), drowning (18 h), suffocation (36 h), and hypothermia (95 h). CONCLUSION The lung hypostasis and the appearance of pleural space fluid collection presented differently in individual causes of death and depending on the postmortem time.

TLR4 stimulation and corticosteroid interactively induce osteonecrosis of the femoral head in rat

We previously reported that a toll‐like receptor 4 signaling contributes to the development of osteonecrosis of the femoral head. Also, oxidative stress is suggested to be one of the possible pathogenesis of osteonecrosis of the femoral head. A recent study showed that toll‐like receptor 4 signaling leads to oxidative stress. The aim of the present study was to evaluate whether toll‐like receptor 4 stimulation and subsequent corticosteroid treatment lead to the development of osteonecrosis of the femoral head in rat, and oxidative stress is associated with it. Male Wistar rats were randomly divided into four treatment groups: Saline + Saline, Saline + Methylprednisolone, Lipopolysaccharide + Saline, Lipopolysaccharide + Methylprednisolone. Osteonecrosis of the femoral head at 14 days after the treatment was observed in 1 of 10 Lipopolysaccharide + Saline, and 5 of 10 Lipopolysaccharide + Methylprednisolone treated rats. However, it was not observed at all in the Saline + Saline and Saline + Methylprednisolone treated groups. Glutathione peroxidase activity in the liver at 1 day after the treatment was significantly increased when treated with lipopolysaccharide. However, methylprednisolone treatment reduced the activity. On the other hand, glutathione peroxidase activity in the femur did not change in any intergroup. In conclusion, the present study showed that toll‐like receptor 4 stimulation by lipopolysaccharide administration strengthen incidence of corticosteroid‐induced osteonecrosis of the femoral head, however, concomitant oxidative stress via toll‐like receptor 4 signaling may not contribute to the development of osteonecrosis of the femoral head in rats.