The expression of a novel stress protein ‘150-kDa oxygen regulated protein’ in sudden infant death

1. Introduction 

Heat shock proteins are induced when cells and tissues are exposed to various stresses such as heat, radiation, ischemia, etc., and such proteins are generally called ‘stress proteins’ [1]. Recently, many of these stress proteins have been identified [1]. The oxygen regulated protein 150-kDa (ORP-150) was identified by Heacock and Southland [2]. Kuwabara et al. [3] and Matsushita et al. [4] clarified that ORP-150 was a stress protein and elucidated its biological role in the central nervous system. According to their studies, ORP-150 is an inducible endoplasmic reticulum (ER) chaperone, and is expressed in a range of pathological situations, such as hypoxic and ischemic brain, atherosclerotic plaques and malignant tumors.

In forensic medicine, sudden unexpected infant death cases have been a major problem [5], [6]. In particular, the pathophysiology of the sudden infant death syndrome (SIDS) has been not well known. Naeye et al. [7], [8], [9] and Rognum et al. [10] proposed that a chronic hypoxic condition might have a close relationship with SIDS. To test this theory, we performed immunohistochemical and morphometric studies on the expression of ORP-150 in the brains of sudden infant death syndrome (SIDS) cases and discussed the pathophysiological significances of ORP-150 in SIDS cases.

2. Materials and methods 

3. Results 

In the H&E staining, significant findings could not be observed in the neurons and the glial cells. In the immunostaining for ORP-150, positive reactions showing a granular pattern were detected in the cytoplasm of the neurons in the cortex (Fig. 1). This result showed that ORP-150 was localized in the ER of the neurons.

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Fig. 1. The expression of ORP-150 in the cerebral cortex. a (×40); and b (×200).

We examined the correlation between infant age and ORP-150 level with Peason's correlation coefficient test. However, there was no correlation between infant age and ORP-150 expression level (r<0.3).

The cluster analysis is a process of clustering objects into groups where the groups are unknown a priori. The clusters are formed in such a way that objects in the same cluster are similar to each other, while members of different clusters are considerably different from each other. Similarity of objects is often measured by some indices of association. In the cluster analysis, the 18 cases were classified into three groups (groups A–C). The mean value and standard deviation of ORP-150 positive cells was in Group A was 66.75±3.44, Group B was 39.50±2.52 and Group C was 16.00±2.92, respectively. We found a relationship between cause of death and mean value of ORP-150 positive cells. Group A was composed of six SIDS cases, Group B of six severe respiratory infectious disease cases such as pneumonia and bronchitis including sepsis, and Group C of five SIDS and one severe respiratory infectious disease (Fig. 2).

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Fig. 2. The relation between the number of ORP-150 positive cells and cause of death as classified by cluster analysis. S: SIDS case, R: death due to severe respiratory infectious disease including sepsis.

4. Discussion 

ORP-150 belongs to the family of heat shock proteins and the protein functions biologically as a molecular chaperone [3]. In eukaryotes, complex protein modifications, such as a disulfide bond and addition to the sugar chain in the ER, are essential for extra-cellular transportation. A great deal of oxygen is spent in the protein modification; therefore, under a condition where oxygen is not fully supplied, immature proteins accumulate at the ER. In order to prevent irreversible changes in these immature proteins, ORP-150 is induced as a molecular chaperone [3]. Tamatani et al. demonstrated that ORP-150 was also expressed on the neurons of infarcted brain. Moreover, they examined the ORP-150 expression in culture human neurons only under the hypoxic condition. The neurons strongly expressed ORP-150 at 4–8 h after hypoxia [13].

In this study, the cerebral cortex in the parieto-temporal lobe was examined. The parieto-temporal lobe is a border area supplied by the anterior cerebral artery and the middle cerebral artery. Therefore, in general, this area is considered to well reflect the hypoxic and ischemic condition. But, significant morphological changes of the neurons and the glial cells could not be detected in the H&E staining. However, the expression of ORP-150 was observed as a granular pattern in the cytoplasm of the neurons, which shows that ORP-150 was localized at the cytoplasm. This expression pattern was consistent with previous studies [4].

Cluster analysis also divided the 11 SIDS cases into two different groups: Group A (66.75±3.44) and Group C (16.00±2.92), meaning high ORP-150 expression group and low group. ORP-150 expression is presumed to reflect the degree of chronic hypoxia. Thus, Group A is considered to last hypoxic condition more than Group C. In general, a hypothesis that SIDS is presumed to be composed of heterogenous concepts such as chronic hypoxia, metabolic disorder, and abnormality of cardiac conduction system has been accepted. In particular, Naye et al. [7], [8], [9] and Rognum et al. [10] proposed that chronic hypoxia might be one factor that contributes to SIDS. Moreover, Takashima et al. [14], [15] and Yamanouchi et al. [16] suggested that the development of synapses in the respiratory center might be impaired in SIDS cases. These studies suggested that immaturity of the neural regulation system in the respiratory movement might contribute to SIDS, and as a result, a chronic hypoxic state is likely to be antecedent. The difference of ORP-150 expression between Group A and C may provide some information to the concepts of SIDS, suggesting that SIDS is not homogenous disease but composed of heterogenous disease concepts. Thus, the intensive ORP-150 expression on brain in the SIDS cases is likely to reflect chronic hypoxic condition in the process to death, which can support the hypothesis that chronic hypoxia is presumed to be one pathogenic factor in SIDS.

The death of five SIDS cases in Group C showing lower positive cell number was presumed to occur immediately or nearly immediately, indicating that the five cases had almost no hypoxic brain damage in the process to the death. In the five SIDS cases, pathogenic factors except for chronic hypoxia may be involved. Thus, in these five cases, a more detailed examination concerning disorders of the circulatory system, and endocrine system is necessary.

In Group B being composed of six severe respiratory infectious disease such as pneumonia and bronchitis including sepsis, the expression of ORP-150 moderately increased, thus suggesting that a transient not chronic hypoxia might last in the process of death. However, only one respiratory infectious disease case was included in Group C (low ORP-150 expression). Although we cannot fully explain the reason, the severity score of respiratory infection in the case was the lowest between seven infectious disease cases.

Finally, it is very difficult to morphologically differentiate SIDS from mechanical asphyxia. In the present study, there were no cases of acute mechanical asphyxia. However, we guess that, in cases of death due to acute mechanical asphyxia, no significant increase in the number of ORP-150 positive cells is likely to occur, because ORP-150 is induced several hours after cerebral ischemia in animal experiments. Although it is needless to say that further study is necessary, immunohistochemical analysis of OPR-150 in the brain may be very useful in differentiating between SIDS and acute asphyxia.