Ultrathin sections of human brain were made and investigated by electron microscope; The fine structures of nerve cells, glia cells, nerve and glia fibres and Nissl's grey were clearly observed. These results were obtained from single section method, but there are many points, which are not yet cleared, the series section method seems to be necessary for the purpose of making them clear.
1) For the purpose of preserving the fine structure of cells, the osmic acid was the best for fixation and then in the order of formalin, Zenker's formalin solution, Bouin's solution, Carnoy's solution, alcohol and aceton. 2) By formalin fixation the ground substance of the nuclei was preserved best. 3) By alcohol fixation the cells were destroyed most severely and tended to make dense and coarse precipitations. 4) Zenker's formalin solution and Bouin's solution gave almost similar figures of fixation, but the former preserved better the fine structures. 5) By Carnoy's solution the fine structures were slightly better preserved than by alcohol. 6) After aceton fixation no good figures were obtained for electronmicrosopy. 7) The fixing solutions except osmic acid were not satisfatory enough by single use, but when they were combined with osmic acid, they became useful to clarify the figures.
1) The main postmortal autolytic changes were melting or agglutination of the fine granules, swelling and contraction of the cells. 2) The specimen for electron microscope should be fixed early as possible. 3) The fine structures were preserved relatively well within 2 hours after death. Therefore the specimens obtained within 2 postmortal hours could be used satisfactorily for the electron microscope. If unavoidable, the specimens obtained until 5 postmortal hours could be used, but the investigation of the fine changes might be difficult. 5) The specimens over 5 hours could not be used for electron microscope.
Using the tissue culture of the bone marrow, I have studied the mechanisms of adrenalin cortisone and ACTH eosinopenia in the bone márrow. The results are as follows: (1) For the mechanism in the bone marrow, adrenalin eosinopenia is based on checking or migratory impossibility of the eosinophil to the bone marrow sinus, and not on the reduced production of the bone marrow. (2) Cortisone eosinopenia is caused from the hypofunction of the eosinophil or from developmental disturbance of the bone marrow parenchyma. (3) Adrenalin or cortisone eosinopenia greatly depends upon its quantitative proportion in the medium. (4) For the direct effect on the bone marrow, ACTH causes the hyperfunction of the eosinophil in a paticular manner but has no influence upon the cell division of the eosinophil. (5) The pseudoeosinophil is free from hypofunctional action and developmental disturbance of adrenalin, cortisone, or ACTH, except the case of high concentration of adrenalin as well as cortisone.
I have studied adrenalin, cortisone and ACTH eosinopenia in hypophysectomized dogs by means of chamber counting. In addition, the bone marrow of hypophysectomized dogs was cultivated in using the plasma of normal and hypophysectomized dogs separately, cortisone was added to each preparate. The results obtained are as follows: (1) Adrenalin eosinopeia is due to its direct influence upon the eosinophil partly through the pituitary body. (2) ACTH acts as glucocorticoid in ACTH eosinopenia. This brings about the similar result to cortisone eosinopenia. But ACTH eosinopenia has no correlation with the function of the pituitary anterior lobe, while cortisone eosinopenia requires the normal function of the anterior lobe. (3) Cortisone (compound E) alone can not cause cortisone eosinopenia but it gives hypofunctional influence and developmental disturbance upon the eosinophil in synergism of other compounds. (4) The normal functions of both the adrenal cortex and pituitary anterior lobe is necessary for eosinopenia by cortisone administration. (5) Cortisone test can be used as a test of pituitary anterior lobe function only in the case with normal adrenocortical function.
Direct effect of adrenalin, cortisone and ACTH on the eosinophilic leucocyte were observed on the standpoint of moving form by the same method described in part (I). The results are as follows: (1) Adrenalin acts directly on the eosinophil and hypofunctionally on its moving form. (2) Cortisone acts directly on the eosinophil and hypofunctionally on its moving form. (3) When ACTH acts directly on the eosinophil, it brings about the hyperfunction on the standpoint of its moving form. These facts add morphological evidence to the change of functions of the eosinophil caused by drugs which has been described in part (1).
Using the culture on a cover slip. I have studied the direct influences of ACTH and cortisone upon the bone marrow obtained from patients with blood diseases. The results are as follows: (1) ACTH shows an excellent effect on the bone marrow of intramedullary blood cell arresting type of hypoplastic anemia but no effect on the other types, especially on panmyelophthisis. (2) For the bone marrow of acute myelogenous leukemia, ACTH and cortisone have a good effect but not for the chronic type. Cortisone has a considerably good effect on monocytic leukemia and lymphocytic leukemia. (3) Cortisone is excellently effective for the bone marrow of Banti's disease. (4) ACTH shows an excellent effect on the bone marrow of agranulocytosis and cortisone shows a good effect.
The epileptie convulsion was caused by local injection of 10% metrazol in the cerebellar cortex and its march was researched. The results were as follows. 1) The convulsion was identical with the cerebral cortical epileptic convulsion and the number of cases in which the march of spasm was observed was quite the same as that of cases in which the convulsion occurred simultaneously in all parts of body. 2) No convulsion was occurred by the stimulation upon the vermis cerebellaris and also it occurred very rarely by that upon the cerebellar nuclei. 3) In cases having the march of spasm caused by stimulation upon Lobus lunatus anterior (L. l. ant.) began in the fore limb, while by stimulation upon L. l. inf. and L. semil. mainly in the hind limb on the same side of stimulation. 4) In cases of cerebellar stimulation, the pathway of the impulse was to be between the both cerebellar hemisphere and both thalami and thus the march of spasm spread from one side of the body to the other side. 5) No march of the cerebellar epileptic convulsion occurred without the cerebral motor cortex. 6) After the removal of both side of the cerebral motor cortex no march occurred but the general convulsion occurred simultaneously. 7) No convulsion occurred by stimulation upon the cerebellar hemisphere after the removal of both thalami or both nuclei lenticulares. 8) The march of convulsion was occurred by a close assimilation of pyramidal and extrapyramidal tracts. It seems that for impulse of convulsion the extrapyramidal tract plays important role, while for start of march the pyramidal tract plays mainly.
After the removal of the thalamus, nucleus caudatus, nucleus lenticularis, superior colliculus, or internal capsule, or after laminar coagulation of the motor cortex, electrical and chemical stimulation was made on the area 4c of the motor cortex ef dogs. The results were as follows: 1) An adversive movement occurred by electrical stimulation and metrazol injection on the area 4c of the cerebral cortex. 2) The adversive movement from the area 4c does not pass through the thalamus, nucleus caudatus, nucleus lenticularis or superior colliculus, but through the direct efferent pathway in the internal capsule. 3) The adversive movement from the area 4c passes through the pyramidal tract.
Author researched the pathways of the adversive movement anatomically on dogs by the Marchi method and Nauta staining method. The results were as follows: One of the first neurons from the area 8 and 6aβ of cerebral cortex terminated in the homolateral ventral nucleus of the thalamus. The other first neurons from the area 5, 7, 19, and 22 terminated in the head and tail of the homolateral caudate nucleus. The second neurons from the ventral nucleus of the thalamus terminated in the homolateral rostal colliculus, and interstitial nucleus. The other second neurons from the caudate nucleus reached the homolateral lentiform nucleus. The third neurons from the lentiform nucleus connected with the rostal colliculus, Darkschewitsch's nucleus, interstitial nucleus, oculomotor and trochlear nuclei of the homolateral side. The neurons from the rostal colliculus connected with the oculomotor, trochlear, abducens, facial, accessory nuclei etc. The neurons from the dentate nucleus passed through the brachium conjunctivum and reached the contralateral ventral nucleus of the thalamus, Darksche witsch's nucleus, interstitial nucleus, oculomotor nucleus and then connected with the descen ding pathways from the cerebral cortex.
Electric stimulation was performed on the motor cortex of the human brain in 30 cases of epileptics and then histological investigation was made on the removed specimen of the brain. The extent of the center for the upper limb was relatively wide and the extent of the center for the lower limb was hardly discovered by electric stimulation. In the center of the upper limb, the extent for fingers was the widest and responded by the lowest voltage, then following order of the wrist, the elbow and the shoulder joint. The area anteriorly close to the Rolandic fissure, even if without any motor response to electric stimulation, had mostly the giant pyramidal cells of Betz, especially the area close to the midline had always bigger Betz cells. The gyri anteriorly distant from the Rolandic fissure, even if with motor response to electric stimulation, hardly had the Betz cells. No Betz cells were observed in the responded area of the area 6 and posterior to the Rolandic fissure. From these results. the theory seems to be not reliable that the motr response caused by electric stimulation is due to the Betz cells.
The same method of investigation as stated in part I was used on 4 cases of the monkey brain (Macaca muletta). 1) The extent of the center for the hind limb responded to electric stimulation was almost as same as or a little narrower than the extent with the Betz cells, while the extent of the center for the fore limb was much wider than the extent with the Betz cells. 2) The extent with motor response first appeared like islands at the threshold voltage, became larger as the voltage increased and then the extents for the upper and lower limbs became doubled, thus making the mixed area. 3) This enlargement of the mixed area by increased stimulation had a tendency to move towards the center for the lower limb. 4) The threshold of stimulation was the lowest at the center for the upper limb, then at that for the lower limb, and the highest at the mixed area. And even in the same center, the threshold was lower at the central part with more Betz cells and higher at the peripheral part with less Betz cells. From these facts, it would appear that the extent of the motor cortex determined physiologically is not always the same as the extent of the distribution of the Betz cells, but is due to the density and sensitivity of the original nerve cells of the pyramidal tract, regardless of the size of the cells.
The changes of the amount of free amino-nitrogen and glutamic acid were investigated in the rabbits brain with repeated convulsions caused by hexogen. The free amino-nitrogen was measured by Van-Slyke-Neil gasmetric apparatus, and the glutamic acid was measured by Warburg gasmetric apparatus with glutamic acid decarboxylase. The results were as follows. Normal Rabbits Brain During Repeated Convulsions 20-24 Hours after Repeated Convulsions Free Amino-nitrogen 0.4553mg/g 0.3006mg/g 0.3324mg/g Glutamic acid 1.5501mg/g 0.9543mg/g 1.2425mg/g The free amino-nitrogen as well as the glutamic acid were the most in the normal brain and the least during the repeated convulsion, and they showed a certain increase at 20-24 hours after the repeated convulsions compared with those during the convulsions, but still apart from the normal. Glycolysis was depressed by sodium glutamate in the normal rabbits brain which had enough glutamic acid, but the glycolysis was rather accelerated by sodium glutamate in the rabbits brain which had repeated convulsions and thus scarce glutamic acid, when the sodium glutamate was added to the irrigating fluid of the vessels in the brain and then the glycolysis was measured in each irrigating fluid before and after irrigation. Therefore, it could be concluded that sodium glutamate has the effect of regulation to keep the normal and moderate glycolysis in the brain.
As stated in part 1. the factors of convulsions have also influence on tissue respiration of the brain. of which the main part is played by glycolysis. This experiment was performed for the purpose of investigating the effect of glutamic acid on glycolysis in rabbits brain with repeated convulsions caused by hexogen and these substances of 1) glucose, 2) glucose and glutamic acid, 3) sodium pyruvate 4) sodium pyruvate and glutamic acid were added to the irrigating fluid of the vessels in the brain. The results were as follows: In the experiment with added glucose, glycolysis was depressed in the brain with convnlsions compared with the normal and was accelerated when glutamic acid was added. In the experiment with added sodium pyruvates, the changes caused by the diluted blood, i.e. the medium were so striking that definite conclusion seemed to be hardly obtained.
It is well-known fact that we can see the cross-immunization with guinea pigs between R. prowazeki and R. mooseri. But we cannot find any reports concerning the experiments about the grades of immunization by R. prowazeki or R. mooseri. The author took the method to immunize the guinea pigs through the subcutaneous route. The antigens used were formol vaccines made from Citellus mongolicus infected with R. prowazeki and R. mooseri, Cox-Craigies vaccine and a small, but enough amount of viruses to cause latent infection. Challenge tests against the immunized guinea pigs were performed through intracardial, intratesticular and subcutaneous route. The guinea pigs, immunized by various kinds of antigen, were challenged through various routes by the rickettsial strains. The results of this cross-immunization were as follows: 1. Formol vaccine of R. prowazeki proved to have the protective activity against challenges by homologous and heterologous rickettsial strains regardless of the ways of their passage. 2. Formol vaccine of R. mooseri proved to have complete protective activity against homologous strain, and also have partial protective activity against heterologous strain which had been generated through intraperitoneal route, but only a little protective activity against heterologous strain which had been generated through intracardial route. 3. Concerning the protective activities, induced from latent infection of R. prowazeki and R. mooseri, the guinea pigs immunized by R. prowazeki showed better protective activity against homologous strain than against heterologous one. 4. The protective activity induced from Rickettsia prowazeki had a tendency to be inferior against R. prowazeki strain, which had been generated successively through intracardial route, to the strain generated through the other routes. 5. Conclusively, to see the protective activities of the immunized guinea pigs against rickettsial strains used for challenge, we must know and regard about the strains how had been their successive generation made and what routes had been taken to innoculate.
The differences between R. prowazeki and R. mooseri have been, so far, discussed from their epidemiological traits, clinical signs, affinity for tunica vaginalis of guinea pigs or rats, affinity toward endothelial cells of blood vessels, structure of antigen, and antigenicity of complement fixation test, In recent time, the way of thinking about the differences between these rickettsiae tends to place great importance on complement fixation test in place of affinity toward tunica vaginalis or endothelial cells of blood vessels of guninea pigs. In order to clear the questionable points to differentiate the two types of rickettsia, the author has studied about the affinity of the rickettsia toward tunica vaginalis of guinea pigs and has considered again about the phenonena occurring in scrota of guinea pigs. Heart bloods of guinea pigs infected with R. prowazeki and R. mooseri were mixed, and this mixed blood was used as a starting-material for successive generation. The author found that by the successive generation to 10 generations through intraperitoneal route suiting to R. mooseri or through intracardial route suiting to R. prowazeki, the rickettsial strains of the original mixed blood could be isolated to each type of rickettsiae on the ground of complement fixation test about the isolated strains. It is not too much to say that scrotal phenomena caused by R. mooseri is, as expected, very important sign to differentiate R. mooseri from R. prowazeki.