Chromatin Ultrastructural Abnormalities in Leukocytes,as Peripheral Markers of Bipolar Patients

This study investigated the ultrastructural conformation changes of the chromatin in blood leukocytes of bipolar patients, versus normal controls, by using the phosphotungstic acid-hematoxylin (PTAH) block-staining method, modified for electron microscopy, and the immunohistochemical localization of the histone H1, by the immunogold method. These two methods are basically complementary. If histone H1 immunolabeling is used, it shows that the immunogold labeling on chromatin is different in the three phases of the illness, i.e., high in normothymia and low in depression as well as in mania. However, in this particular tissue fixation (4% paraformaldehyde-1% glutaraldehyde in 0,1 M phosphate buffer), the heterochromatin in the nuclei remains identical in the three phases of the illness. On the other hand, the PTAH method shows exactly the area of electron-lucent condensed chromatin, separate from the area of electron-dense, decondensed, chromatin. The present data confirmed that both the clinical state of depression as well as that of mania display activated lymphocytes and neutrophils with their characteristic relaxed de-condensed chromatin. On the contrary, the state of normothymia shows a reversion to the condensed state of the chromatin, as it is observed in the leukocytes of the normal controls. The ultrastructural conformations of the chromatin, revealed by the PTAH method, in combination with the histone H1 immunogold labeling, applied in blood leukocytes, supports the use of these two methods, as screening methods of choice in investigating blood biological markers in mental illness.     

Chromatin Alterations in Leukocytes of First-episode Schizophrenic Patients

Studies of peripheral blood leukocytes of schizophrenic patients have shown in electron microscopy (EM) that decondensation of the chromatin constitutes a biological marker indicating increased genomic expression. Since this increase depends on chromatin relaxation by dissociation of lysine-rich histone H1 from nucleosomes, with exposure of arginine residues of core histones, the ratio of arginine to lysine residues in each nucleus represents a reliable measure of activation. Lysine- and arginine-rich proteins are demonstrable in light microscopy (LM), differentially, as yellow and black, respectively, with the ammoniacal silver reaction (ASR). Application of ASR on leukocyte pellets before they are dehydrated and embedded in epoxy resins gives reliable results in semithin sections. In thin sections the ASR method localizes only the amino acid arginine by forming deposits of electron-opaque particles, visualized in the EM. Leukocytes of 12 first-episode schizophrenic patients and 5 controls were used. Light micrographs of the semithin sections were inserted in a personal computer. The percentage of lysine and arginine was measured in 300 nuclei per subject. Morphometry showed that lymphocytes of schizophrenic patients have increased ratios of arginine to lysine, compared to controls, indicating activation; neutrophils of the patients have even a higher ratio, indicating an abnormal condition of the genome. Chromatin conformational changes are also evident by phosphotungstic acid hematoxylin (PTAH) block staining, which reveals condensed chromatin as an electron-lucent area in the nuclei, and decondensed chromatin as an electron-dense area. Because decondensed chromatin is a biological marker of schizophrenia, the efficacy of these methods to demonstrate this particular state offers a tool for early diagnosis, since first-episode schizophrenic patients have a better prognosis when treatment is started promptly, at the beginning of the disease.     

The perforating disorders

This article reviews the diseases that may show epidermal perforation as a histologic feature. Many of these represent examples of transepithelial elimination (TEE), a mechanism by which the skin rids itself of abnormal substances. After a review of disorders in which perforation is an occasional finding, four diseases that have been considered essential perforating disorders are discussed: elastosis perforans serpiginosa (EPS), reactive perforating collagenosis (RPC), perforating folliculitis (PF), and Kyrle 's disease (KD). A review of the literature, including recent reports of perforating diseases associated with chronic renal failure, suggests that there may be considerable clinical and histologic overlap among PF, KD, and the adult form of "perforating collagenosis." A working classification for the perforating disorders is suggested.     

特殊染色在非临床药物安全性评价中的应用

病理学诊断中特殊染色具有重要的应用价值,通过长期的改进与创新特殊染色能够有针对性地对组织成分进行染色,使组织病理学评价更加完善精确。非临床药物评价中常用的特殊染色法有结缔组织染色的Masson三色染色法、肌肉染色的磷钨酸苏木素染色法、糖原染色的过碘雪夫染色法、脂质类染色的油红O染色法、色素类染色的Masson-Fontana染色法,以及神经组织染色和病理沉着物染色等,综述特殊染色的主要内容及应用中注意事项。     

Activated inflammatory cells participate in thrombus size through tissue factor and plasminogen activator inhibitor-1 in acute coronary syndrome: Immunohistochemical analysis

Introduction

Recent studies have suggested that circulating inflammatory cells augment the growth of thrombus in acute coronary syndrome (ACS). We therefore immunohistochemically analyzed thrombi in aspirates obtained from patients immediately after the onset of ACS.

Materials and Methods

Two hundred twenty samples were studied. Total thrombus area, white thrombus area, and red thrombus area were measured. As antibodies in immunohistochemical staining, myeloperoxidase (MPO), CD66b, CD68, p-selectin, tissue factor (TF) and PAI-1were employed respectively.

Results

The ratios of areas of red and white thrombi correlated with whole sample areas of enlarged thrombi (r = 0.48, p < 0.001). The immunohistochemical findings revealed granulocytes and macrophages aggregated around p-selectin-positive platelets that shared the boundary between white and red thrombi, a region where MPO and CD66b expression was abundant in neutrophils. The ratios (%) of MPO- and CD66b-positive cells significantly correlated with whole sample areas (r = 0.50; p < 0.001 and r = 0.49; p < 0.001, respectively). Neutrophils and macrophages within thrombi were positive for TF and PAI-1. Along the boundary between red and white thrombi, TF and PAI-1 positivity coincided with MPO-, CD66b- and CD68-positive cells. The ratios of cells positive for both TF and PAI-1 in this area significantly correlated with the whole sample area (r = 0.43, p < 0.001 and r = 0.60, p < 0.001, respectively).

Conclusions

These results suggested that enhanced activation of peripheral neutrophils together with increased TF and PAI-1 expression might comprise a considerable portion of thrombus enlargement.     

Receptors for leptin and estrogen in the subcommissural organ of rabbits are differentially modulated by fasting

In rabbits, the fasting-dependent reduction of LH secretion is likely mediated by leptin and estrogens via receptors in the brain. For the first time, using immunohistochemistry, the presence and regulation of receptors for leptin (Ob-R) and estradiol-17beta subtype alpha (ERalpha) were studied in the subcommissural organ (SCO) of rabbits, which were fed either ad libitum (control) or fasted for 48 h (treated) to verify whether this brain structure is a potential site of integration for metabolism and reproduction. In control rabbits, the cytoplasm of glial cells lining the SCO evidenced strong Ob-R immunoreactivity, whereas both ependymal and hypendymal cells of this glandular-like structure were negative. The Ob-R positive glial cells were identified as fibrous astrocytes using the phosphotungstic acid-hematoxylin histochemical (PTAH) and glial fibrillary acidic protein (GFAP) immunohistochemical techniques. ERalpha immunoreactive nuclei were detectable exclusively in the specialized cells forming the SCO, whereas surrounding astrocytes and neurons were negative. Compared to controls, in fasted rabbits, the staining of Ob-R immunoreaction was reduced in the cytoplasm of positive astrocytes, but greatly enhanced in plasma membranes, whereas the number of ERalpha immunoreactive SCO cells was increased (13.2+/-2.7 vs. 5.2+/-2.0, P<0.01). Ependymal cells lining the third ventricle were negative for both Ob-R and ERalpha. Our results indicate, although indirectly, that the SCO, together with the astrocytes in close contact with this structure, is a likely target for nutritional and gonadal signals carried by leptin and estrogens, suggesting that these specialized glial cells may regulate reproduction and metabolism through mechanisms still unknown.     

Purified thromboplastin causes haemostatic abnormalities but not overt DIC in an experimental rabbit model

Validation of animal models of disseminated intravascular coagulation (DIC) to human DIC is crucial in order to translate findings in research models to treatment modalities for DIC in humans. ISTH classifications of overt and non-overt human DIC have proven to have a high diagnostic accuracy, and we have previously established a rabbit model of non-overt DIC based on the ISTH classification of non-overt DIC. In this rabbit model, we used purified rabbit brain thromboplastin to induce DIC and test applicability of ISTH classifications of overt human DIC.Cardiovascular and haematological parameters from rabbits, either saline-injected or administered a 2.5 mg thromboplastin/kg bolus and a 15 minutes 1.25 mg thromboplastin/kg infusion, were determined at four time points over a 90 minute period. All groups of rabbits were scored at each time point according to the ISTH classifications of overt DIC.Despite the fact that injection of purified thromboplastin resulted in decreased platelet count, increased prothrombin time, activated partial thromboplastin time, level of thrombin-antithrombin complexes and fibrin degradation products, and pulmonary micro-thrombosis, none of the rabbits were diagnosed as having overt DIC according to ISTH classification.We conclude that purified thromboplastin causes haemostatic abnormalities in the rabbit but this experimental model was not diagnosed as overt DIC.     

Enzyme histochemical changes after transection or hemisection of the spinal cord of the rat

The spinal cords of 84 young adult female rats were transected or hemisected at T7 to T8 and the animals autopsied at intervals from 6 h to 14 months postoperatively. Frozen sections of the unfixed spinal cord on either side of the lesion were prepared for enzyme histochemistry, immunocytochemistry, and histology. The most striking enzymatic alterations and their physiological implications were: (i) (Na⁺---K⁺)-activated ATPase activity decreased in axons of the gray and white matter within 6 h after spinal transection and did not return subsequently, whereas the decrease in activity that occurred contralateral to a hemisection was transient. The decreased activity occurred so promptly as to suggest possible roles in the genesis of the initial flaccid paralysis (spinal shock) in the spinal animal and in the temporary paraplegia seen after subtotal spinal injury. (ii) During the first week postoperatively, many axons in the white matter developed large swellings or small varicosities that reacted strongly only for enzymes normally present in the neuronal perikaryon (e.g., AChE, acid phosphatase, NADH-diaphorase, and G6PDH). This histopathological reaction gradually spread rostrally and caudally from the site of injury, but it disappeared as axonal degeneration supervened. (iii) Within 7 days after spinal transection, many neuronal perikarya were chromatolytic and exhibited decreased AChE activity but normal or increased NADH-diaphorase activity. This response is similar to that seen in the cell bodies of regenerating peripheral axons where anabolic processes are favored over neurotransmission-related functions. (iv) Increased cellularity of the spinal parenchyma adjacent to the lesion resulted largely from the proliferation and hypertrophy of astrocytes. These hypertrophied cells, whose identity was confirmed by GFAP immunocytochemistry, reacted with marked intensity for NADH-diaphorase, G6PDH, and Gly3PDH. Such enzyme changes, characteristic of increased protein turnover, indicate that experimental attempts to control gliosis (e.g., by reducing protein turnover or by other means) could be effectively monitored by enzyme histochemistry.     

Astroglial reaction in the gray matter lumbar segments after midthoracic transection of the adult rat spinal cord

Previous studies of cordotomized rats revealed a glial reaction in the gray matter of the spinal cord at sites remote from the lesion, and the present study was done to explore this phenomenon further. Seventy-five young adult female rats were cordotomized and 10 hemicordotomized, both operations at T5. Between 1 and 28 days postoperatively, histologic sections of thoracic and lumbar segments stained by phosphotungstic acid hematoxylin (PTAH, pH 2.37), by periodic acid Schiffdimedon (PAS-D) or by an immunocytochemical method for glial fibrillar acidic protein (GFAP) revealed histological changes as follows: PTAH staining showed that astroglia in thoracic and lumbar regions of the cordotomized rats possessed a swollen, pink-staining cytoplasm and enlarged, thick, dark blue-staining fibrous processes. This response, first noted within 4 days, had intensified by 7 days and was maximal at 14 to 17 days postoperatively. By 28 days, the reaction had diminished but was still readily detectable. The more specific GFAP staining procedure confirmed that the reactive cells were astrocytes and demonstrated that their fibrillar density had increased. The PAS-D reaction revealed glycogen accumulation in glia of the lumbar gray matter within 2 days; this response intensified by 4 days and diminished to normal by 14 days. This reaction was largely concentrated in the perivascular end feet of astroglia, but also appeared in conjunction with perineuronal astroglia. The site of glial reactivity included both dorsal and ventral horns and was particularly noticed in the gray matter surrounding the central canal. In the hemicordotomized rats, the thoracic and lumbar glia response was much more pronounced ipsilaterally than contralaterally. These results support the interpretation that an astroglial response, involving hypertrophy, fibrillogenesis, and glycogen accumulation, occurs in response to degenerating nerve fibers caudal to sites of spinal cord injury.