Axonal regrowth under release of myelin-associated glycoprotein: Chemotaxis by pioneer Schwann cells and Cajal's gigantic clubs.

Myelin-associated glycoprotein (MAG), released from pre-degenerated distal nerves following axotomy, blocks the regrowth of sprouts and naked axons. Ensheathed axons, however, continue to elongate and reach MAG-releasing distal nerves. To determine the regenerative mechanism of ensheathed axons without navigators of axonal growth cones by the film model method, we inserted a MAG-releasing distal nerve segment treated with liquid nitrogen (N2DS) between two films, facing the proximal end of the common peroneal nerves in mice transected four days earlier for axons to become ensheathed. On the third post-operative day (Day 3), axon fascicles, subjected to silver staining, extended toward N2DS but with few branches, forming terminal swellings called Cajal's gigantic clubs (CGCs) that are filled with axonal growth cones. Filter paper wetted with either 250 pg/ml MAG or N2DS showed the same configurations when inserted between the two films. This effect was lost following anti-MAG treatment; fascicles strayed near the parent nerve with numerous branches, formed a net of axons, and tapered towards thin tips at their ends, just like controls without N2DS. Schwann cell bundles on Day 3, detected with anti-S100, formed sheaths of CGCs at their ends, and connected to pioneer Schwann cells (pSCs). To analyze the physiology of Schwann cells, independent of axons, the parent nerve transected four days prior was crushed. On Day 2, with pSCs ahead, Schwann cell bundles extended towards N2DS. On Day 4, main bundles regressed, leaving pSCs motionless. Thus, MAG is a candidate chemoattractant for both pSCs and CGCs.

Study of the association between an anomalous superior vena cava and horseshoe kidney.

BACKGROUND: The incidence of inferior vena cava anomalies in patients with horseshoe kidney is higher than that reported in the general population. As far as we know, no studies have reported the incidence and variations of superior vena cava (SVC) anomalies using multidetector-row computed tomography (MDCT) in patients with horseshoe kidney. METHODS AND RESULTS: Using MDCT, 71 patients with a horseshoe kidney (group A: 45 males, 26 females; mean age, 60.1 ± 10.2 years) and 2,292 patients without a horseshoe kidney (group B: 1,385 males, 907 females; mean age, 61.1 ± 13.5 years) were retrospectively evaluated for the incidence and variations of SVC anomalies, and the incidence of an anomalous SVC was compared between groups. An anomalous SVC was identified in 3 group A patients (4.2%) (double SVC, n=2; persistent left SVC without a right SVC, n=1) and 5 group B patients (0.22%) (double SVC, n=3; persistent left SVC without a right SVC, n=2). MDCT revealed a significantly higher incidence of anomalous SVC in patients with a horseshoe kidney than in those without a horseshoe kidney (P<0.001). CONCLUSIONS: Patients with horseshoe kidney frequently have an anomalous SVC. Although the incidence of horseshoe kidney is related in some way to that of an anomalous SVC, the reasons for their coexistence remain unclear.

Investigating the histological and structural properties of tendon gel as an artificial biomaterial using the film model method in rabbits.

PURPOSE: This study aimed to evaluate the properties of tendon gel by investigating the histological and structural differences among tendon gels under different preservation periods using a rabbit model. METHODS: Forty mature female rabbits were divided into four groups, each containing ten rabbits, on the basis of in-vivo preservation periods of tendon gels (3, 5, 10, and 15 days). We created the Achilles tendon rupture models using the film model method to obtain tendon gels. Tensile stress was applied to the tendon gel to promote maturation. Histological and structural evaluations of the tendon gel were performed before and after applying the tensile force, and the results obtained from the four groups were compared. RESULTS: Although the day-3 and day-5 tendon gels before applying tensile stress were histologically more immature than the day-10 and day-15 gels, type I collagen fibers equivalent to those of normal tendons were observed in all groups after the tensile process. Based on the surface and molecular structural evaluations, the day-3 tendon gels after the tensile process were molecularly cross-linked, and thick collagen fibers similar to those present in normal tendons were observed. Structural maturation observed in the day-3 tendon gels caused by traction was hardly observed in the day-5, -10, and -15 tendon gels. CONCLUSIONS: The day-3 tendon gel had the highest regenerative potential to become a normal tendon by applying a traction force.

Anatomical study of the external carotid artery and its branches for administration of superselective intra-arterial chemotherapy via the superficial temporal artery.

BACKGROUND: The branching patterns of the external carotid artery vary among individuals, and consideration of the proximity of nerves is important during catheter insertion in superselective intra-arterial infusion via the superficial temporal artery. We aimed to evaluate the anatomy of the external carotid artery and its surrounding nerves for safe and accurate administration of superselective intra-arterial chemotherapy via the superficial temporal artery. METHODS: We analyzed the external carotid artery and its branches morphometrically in 28 Japanese cadavers (56 sides). RESULTS: Vascular tortuosity in the preauricular region of the catheter insertion site was observed in 42.9% of the sides; the main trunk of the external carotid artery was excessively tortuous in 25.0% of the sides, primarily in the preparotid region. Faciolingual and superior thyrolingual trunks were observed in 28.6 and 1.8% of the sides, respectively. The superior thyroid, lingual, facial, occipital, and maxillary arteries branched from the external carotid artery above the carotid bifurcation in 41.1% of the sides. The mean distance between the insertion site and maxillary artery was 39.5 mm, indicating the extent of catheter insertion. The auriculotemporal nerve was observed near the superficial temporal artery in the preauricular region in 44.6% of the sides; however, the clearly identifiable nerves in the exposed area were difficult to avoid. CONCLUSION: Because of the branching variations observed in individuals and sides, preoperative angiography is extremely important for avoiding complications.

[Effectiveness of combination learning of basic problem and free clinical topics in lecturing for lower grade students in pharmacy school].

To help pharmacy students develop applied knowledge concerning clinical diseases and the ability to communicate with patients (and medical staff), we have introduced a new improved program for first and second year pharmaceutical students. This new program involves a 10-20 minute presentation of a clinical disease and clinical case by the students after regular lectures. Our new program may be useful for a 6-year pharmacy education in order to produce pharmacy students who have: 1) wide clinical knowledge, 2) a better basis for understanding advanced subjects such as pharmacology, drug therapeutics and pathologic physiology that are taken in the upper grades, and 3) practical training at medical institutions. In addition, a pamphlet produced as part of the student presentation become reference data when students in the following year study the same topic or teachers of other professional subjects attempt a similar program.

Evaluation of dynamic change in regenerated tendons in a mouse model.

BACKGROUND: Using the film model method, the process whereby a substance called tendon gel is secreted from transected tendon ends and changed into a tendon after application of a traction force is known. The objective of this study was to investigate the association between mechanical properties in the early stages of tendon regeneration and time by using the film model method. METHOD: Adult male ddY mice, closed colony mice established and maintained in Japan, were prepared for each experimental group. The study animals were 30 mice and were divided into three groups of 10 mice each. Ten specimens of tendon gel secreted from the transected tendon ends were collected on days 10, 15, and 20 postoperatively. While a traction force of 0.00245 N was applied to these specimens, the process of tendon gel changing into a tendon was video recorded for 24 h, and the length of extension was measured over time. Regenerated tendons were stained with hematoxylin and eosin for histological examination. Healing site was studied histologically according to the our maturity score with reference to the Bonar's scale. RESULTS: The day 10 specimens gradually stretched for 12 h after the start of pulling and transformed into tendons. In contrast, the day 15 and 20 specimens stretched immediately after the start of pulling and transformed into tendons. The day 10 specimens stretched significantly more than the day 15 and 20 specimens (mechanical strain; 0.43 ± 0.26%, 0.03 ± 0.02%, and 0.03 ± 0.01%, respectively)Statistically significant differences were observed in the day 10 specimens than in the day 15 and 20 specimens. (P < 0.017). Using our maturity scores, the day 15 and 20 specimens were more mature than the day 10 specimens. (1.6 ± 0.68, 3.9 ± 0.54, and 4.8 ± 0.64, respectively) Statistically significant differences were observed in the day 10 specimens than in the day 15 and 20 specimens (P < 0.017). CONCLUSION: Tendon gel physiologically and histologically matures on or after day 15 and becomes stronger dynamically in mechanical strength after day 15 than after day 10.

Appropriate Tensile Mode and Timing of Applying Tension to Promote Tendon Gel Regeneration.

"Tendon gel" secreted from a parent tendon is regenerated for tendon repair by applying tension. However, the details of the tensile stimulus have not been clarified. This study aimed to evaluate an appropriate tensile stimulus mode and the optimal timing of applying tension to promote tendon gel regeneration. Tendon gel was prepared using a film model method in mice and was preserved in vivo for 3, 5, and 10 days. Unlike tendon gel on day 3 or day 5, a fibrous structure developed in the tendon gel on day 10 when tension was applied. Infrared spectroscopy revealed that characteristic peaks appearing for the tendon gel on days 3 and 5 disappeared on day 10. Disappearance of the peaks indicated maturity of the tendon gel, and it showed the optimal timing for tension application to the tendon gel. The effect of tensile load on tendon gel preserved for 10 days was investigated using a tensile test, a creep test, or a cycle test. In the tensile test, tendon gel was elongated into a thin cord of collagen fibers with an increase in stress, and the maximum diameter of the collagen fiber was approximately 50 times larger than that in the normal Achilles tendon of mice. The results suggest that the diameter of the oriented collagen fiber is controllable by adjusting the applied load and the time in mature tendon gel.

[Anatomy education in medical and dental schools in Japan].

We studied the anatomy education and the view of anatomy professors on it in medical and dental schools in Japan. In most schools anatomy is taught in the second year. In medical schools, the systematic education separating macroscopic and microscopic anatomy is prevalent. Although the tutorial system has been introduced in 80% of medical schools, its introduction into anatomy education has remained at 30%. The tutorial system is regarded to be more effective by engaged professors than non-engaged. Some kinds of clinical anatomy education have been introduced in half of the medical schools surveyed. In dental schools, on the other hand, macroscopic and microscopic anatomy tend to be taught in combination. One third of the dental schools have introduced clinical anatomy but few schools have a tutorial system. The overwhelming majority of professors are evaluated by students and have regarded the evaluation useful for improving their teaching. They also have thought that the questionnaire and the timing of the evaluation must be considered carefully, and that the evaluation should not be directly used for purposes other than the improvement of education. We have made the proposals for further improvement in anatomy education based upon this study.

Rapid appearance of pathological changes of neurons and glia cells in the cerebellum of microsphere-embolized rats.

Neuropathological changes in the cerebellar cortex of microsphere-embolized rats were studied at 30 min and 3 h after the embolism. Necrotic processes including a sponge-like vacuolation in the molecular layer, a vague outline of some Purkinje cells, and a few pyknotic granule cells having small and dark profiles were identified at sometime between 30 min and 3 h after microsphere-induced embolism in Nissl staining. Glial fibrillary acidic protein staining shows an apparent reduction in the number of Bergmann glial processes in some of the areas where there was necrosis of the molecular layer and poor astroglia processes in the areas subjacent to the pyknotic granule cells. These data demonstrate that within a short time, microsphere-induced cerebral ischemia produces necrosis of cerebellar neurons (i.e. Purkinje and granule cells) and changes in cerebellar glia cells (i.e. Bergmann and astroglia cells), and that these neuropathological changes are secondary phenomenon caused by microsphere blockage of cerebellar blood flow.

Mechanisms of collagen fibril alignment in tendon injury: from tendon regeneration to artificial tendon.

The process by which collagen fibrils are aligned following tendon injury remains unknown. Therefore, we analyzed the process of tendon regeneration by transmission electron microscopy, using a film model method. In mice, the Achilles tendon of medial head was transected. On day 3, after only the proximal end of the transected tendon was placed on film and kept in vivo, a translucent substance containing granules, called tendon gel, was secreted. On day 5, the granules assembled in a loose (L) layer, and coalesced tightly in a dense (D) layer, forming an L-D-L layered pattern. On day 10, granules showed high electron density in H layers, which developed into D-H-D layers on day 13. The distal end was placed on film to face the proximal end. On day 10, the tendon gel showed a D-H-D layer pattern. On day 11, mechanical stress from muscular constriction changed the tendon gel to aligned collagen fibrils (6 ± 2 nm in diameter). Thereafter, the diameter of the fibrils increased. Tendon gel harvested on day 5 or day 10 was pulled manually or by hanging weights (about 0.6 MPa). Aligned collagen fibrils (32 ± 7 nm in diameter) were created by traction using tendon gel harvested on day 10.

Hyaluronan tetrasaccharide promotes regeneration of peripheral nerve: in vivo analysis by film model method.

Hyaluronan (HA) is known to inhibit neurons from regenerating in the central nervous system. However, hyaluronan tetrasaccharide (HA4) was found in in vitro experiments to promote outgrowth of neurons. To investigate the promotion by HA4 of nerve regeneration in vivo, we analyzed outgrowth of regenerating axons treated with HA4, using a film model method. After the common peroneal nerve in mice was transected, the proximal end of cut nerve was placed on a sheet of thin plastic film, immersed in several drops of HA4 solution, covered with another sheet of film, and then kept in vivo. Six hours after the procedure, terminal sprouts had grown out from ending bulbs formed at the cut end of parent nerve administered with HA4 solution 100 or 1000 µg/mL, while no sprouts were observed in groups treated with 10 µg/mL of HA4 or in controls. On the 2nd day after axotomy (day 2), many regenerating axons in the group treated with 100 µg/mL of HA4 extended onto the flat film for a longer distance than those treated with 1000 µg/mL of HA4 and controls. With the optimal dose of HA4 (100 µg/mL), axonal outgrowth was significantly (p<0.01) greater than that in controls at each time point. Schwann cells appeared migrating from parent nerve onto the film from day 3 as well as in controls. Thus, enhanced outgrowth of regenerating axons and normal behavior of migratory Schwann cells suggested that HA4 promoted regeneration of neurons without the mediation of Schwann cells.

Time course and sequence of pathological changes in the cerebellum of microsphere-embolized rats.

Ischemia is a major cause of damage to the central nervous system as a consequence of stroke or trauma. Here, we analyzed with high temporal resolution the time course of pathological changes in the neurons (granule and Purkinje cells) and glia (Bergmann and astroglia cells) in the cerebellar cortex and white matter. The period studied ranged from 30 min to 7 days after a microsphere-induced embolism used as a model of stroke and multi-infarct dementia. Some pathological changes in the neurons in the cerebellar cortex were identified early, that is, beginning at 3 h after the microsphere-induced embolism, and glial pathology appeared only later. The pathological changes in the white matter also appeared slightly later, that is, 6 h after embolism and were less pronounced than those in the cerebellar cortex. This suggests that neuronal pathology is induced more rapidly and/or more easily than the glial pathology. In addition, BrdU staining shows that cell proliferation is limited to a 1-day period beginning about 1 day after the embolism. These data demonstrate that changes after an ischemic lesion of the cerebellum proceeds from upper cerebellar cortex to deeper cerebellar cortex or white matter and also that microsphere-induced changes proceed from neuronal to glial pathology.