Topical Skin Application of Small-Molecule Antiplatelet Agent against Pressure Injury in Rat Models.

Due to prolonged forced positioning, the incidence of intraoperative pressure injuries is high. This study aimed to explore the impact of small-molecule antiplatelet drugs on pressure injuries by locally applying them before an injury occurs. In the first part of this study, water-soluble tracers with different molecular weights were applied to normal and early-stage pressure-injured skin. Through digital cameras, spectrophotometers, and histological observations, the penetration of tracers into the epidermis was clarified. In the second part of this study, a water-soluble antiplatelet drug called Trapidil (molecular weight = 205 Da) was applied to the left side of the back of a rat before, during, and after compression, and the contralateral side served as a non-intervention control group. The differences in pressure injuries between the two groups were observed through a digital camera, an ultraviolet camera, and temperature measurement, and skin circulation and perfusion were assessed via an intravenous injection of Evans Blue. The first part of this study found that water-soluble tracers did not easily penetrate normal skin but could more easily penetrate pressure-damaged skin. The smaller the molecular weight of the tracer, the easier it penetrated the skin. Therefore, in the next step of research, water-soluble drugs with smaller molecular weights should be selected. The second part of this study found that, compared with the control group, the occurrence rates and areas of ulcers were lower, the gray value was higher, and the skin temperature was lower in the Trapidil group (p < 0.05). After the intravenous Evans Blue injection, skin circulation and perfusion in the Trapidil group were found to be better. In conclusion, this study found that the topical skin application of a small-molecule antiplatelet agent may have significant effects against pressure injuries by improving post-decompression ischemia, providing new insights into the prevention and treatment of intraoperative pressure injuries.

Fluctuations in Detection Indicators and Their Significance for the Diagnosis of Early Pressure Injury in Rat Models.

PURPOSE: The aim of this study was to identify the most meaningful diagnostic indicator for distinguishing blanchable erythema (BE) and stage 1 pressure injury (early PI) in an in vivo (rat) model. DESIGN: A prospective case-control design was used to complete a horizontal and vertical comparison of detection indicators during the process of fading of BE or the deterioration of early PI into ulcer in rat models. MATERIALS AND SETTING: The sample comprised 5 hairless rats with 20 injuries, of which 10 were BE and the other 10 were early PI. Data were collected at Nagano College of Nursing in 2020 in Nagano, Japan. METHODS: The BE and PI rat models were established by subjecting the dorsal skin of a hairless rat to compression between 2 neodymium magnets for 45 minutes and 3.45 hours, respectively. The affected skin was observed based on the following: (1) photography, (2) hardness, (3) temperature, (4) moisture, and (5) spectrophotometric (a* value and ultraviolet [UV] reflectance) measurements. All measurements of BE were performed at the beginning to 60 minutes after decompression, and those for early PI were performed until 48 hours after decompression. RESULTS: Multiple BE factors, such as the degree of erythema (macroscopy and a* value), hardness, temperature, and moisture, were found to have unstable fluctuations. Only UV reflectance gradually decreased from 6 hours and decreased significantly at 48 hours after decompression (P = .001 vs 1 hour). In contrast to early PI, erythema in BE obviously faded within 10 minutes. CONCLUSIONS: Study findings indicate that a continuous decrease in UV reflectance can reflect the worsening of hemorrhage in early (stage 1) PI. In contrast, other indicators including photography, skin hardness, temperature, and moisture fluctuated and did not prove predictive for PI progression. The obvious fading of erythema in BE a short time after decompression can be used for clinical observations.

Involvement of membrane palmitoylated protein 2 (MPP2) in the synaptic molecular complex at the mouse cerebellar glomerulus.

We previously reported that the membrane skeletal protein 4.1G in the peripheral nervous system transports membrane palmitoylated protein 6 (MPP6), which interacts with the synaptic scaffolding protein Lin7 and cell adhesion molecule 4 (CADM4) in Schwann cells that form myelin. In the present study, we investigated the localization of and proteins related to MPP2, a highly homologous family protein of MPP6, in the cerebellum of the mouse central nervous system, in which neurons are well organized. Immunostaining for MPP2 was observed at cerebellar glomeruli (CG) in the granular layer after postnatal day 14. Using the high-resolution Airyscan mode of a confocal laser-scanning microscope, MPP2 was detected as a dot pattern and colocalized with CADM1 and Lin7, recognized as small ring/line patterns, as well as with calcium/calmodulin-dependent serine protein kinase (CASK), NMDA glutamate receptor 1 (GluN1), and M-cadherin, recognized as dot patterns, indicating the localization of MPP2 in the excitatory postsynaptic region and adherens junctions of granule cells. An immunoprecipitation analysis revealed that MPP2 formed a molecular complex with CADM1, CASK, M-cadherin, and Lin7. Furthermore, the Lin7 staining pattern showed small rings surrounding mossy fibers in wild-type CG, while it changed to the dot/spot pattern inside small rings detected with CADM1 staining in MPP2-deficient CG. These results indicate that MPP2 influences the distribution of Lin7 to synaptic cell membranes at postsynaptic regions in granule cells at CG, at which electric signals enter the cerebellum.

Predicting the different progressions of early pressure injury by ultraviolet photography in rat models.

Early pressure injury (PI) can result in either spontaneous healing (SH) or deterioration into ulcer (DU). However, determining whether PI will progress into SH or DU on the basis of non-blanchable erythema only is difficult. In this study, we constructed two animal PI models to mimic SH and DU injuries and observed haemorrhage by using ultraviolet (UV) photography to develop potential clinical indicators for predicting the progression of early PI. Macroscopy, UV photography, and skin temperature observations were obtained. In the SH group, macroscopic observation showed the erythema was obvious at 0.5 hours after decompression and faded gradually had almost disappeared at 72 hours. In the DU group, the erythema persisted, and an erosion appeared at 24 hours after decompression and expanded at 36 hours. The erythema developed into an obvious ulcer at 48 hours and enlarged at 72 hours. The obvious ulcer found at 48 hours through macroscopic observation was clearly visible at 36 hours with UV photography, and a significant difference in grey values between the two groups was found at as early as 18 hours (P < .05). This study provided evidence showing that UV photography can predict the different progression stages of early PI. Additionally, when combined with the transparent disc method, UV photography also can be used to identify the circulatory disorders of early PI, such as haemorrhage or hyperaemia and even congestion.

The membrane palmitoylated protein, MPP6, is involved in myelin formation in the mouse peripheral nervous system.

A membrane skeletal molecular complex, protein 4.1G-membrane palmitoylated protein 6 (MPP6)-Lin7-cell adhesion molecule 4 (CADM4), is incorporated in Schwann cells, especially in Schmidt-Lanterman incisures (SLIs), in the mouse peripheral nervous system (PNS). MPP6, Lin7, and CADM4 are transported to SLIs by 4.1G. In this study, we created MPP6-deficient mice and evaluated myelin structure and MPP6 protein complexes. In SLIs in MPP6-deficient nerves, Lin7 was rarely detected by immunohistochemistry and western blotting, but the localization and amount of CADM4 and 4.1G were not altered. Motor activity was not significantly impaired in a tail-suspension test, but the sciatic nerves of MPP6-deficient mice had thicker myelin in internodes by electron microscopy compared to that of wild-type mice. These results indicate that the MPP6-Lin7 complex regulates myelin formation.

Scaffold protein Lin7 family in membrane skeletal protein complex in mouse seminiferous tubules.

The membrane skeletal complex, protein 4.1G-membrane palmitoylated protein 6 (MPP6), is localized in spermatogonia and early spermatocytes of mouse seminiferous tubules. In this study, we investigated the Lin7 family of scaffolding proteins, which interact with MPP6. By immunohistochemistry, Lin7a and Lin7c were localized in germ cells, and Lin7c had especially strong staining in spermatogonia and early spermatocytes, characterized by staging of seminiferous tubules. By immunoelectron microscopy, Lin7 localization appeared under cell membranes in germ cells. The Lin7 staining pattern in seminiferous tubules was partially similar to that of 4.1G, cell adhesion molecule 1 (CADM1), and melanoma cell adhesion molecule (MCAM). Lin7-positive cells included type A spermatogonia, as revealed by double staining for Lin28a. Lin7 staining became weaker in MPP6-deficient mice by immunohistochemistry and western blotting, indicating that MPP6 transports and maintains Lin7 in germ cells. The histology of seminiferous tubules was unchanged in MPP6-deficient mice compared to that of wild-type mice. In cultured spermatogonial stem cells maintained with glial cell line-derived neurotropic factor (GDNF), Lin7 was clearly expressed and immunolocalized along cell membranes, especially at cell-cell junctions. Thus, Lin7 protein is expressed in germ cells, and Lin7, particularly Lin7c, is a useful marker for early spermatogenesis.

Structures and Molecular Composition of Schmidt-Lanterman Incisures.

Schmidt-Lanterman incisure (SLI) is a circular-truncated cone shape in the myelin internode that is a specific feature of myelinated nerve fibers formed in Schwann cells in the peripheral nervous system (PNS). The SLI circular-truncated cones elongate like spring at the narrow sites of beaded appearance nerve fibers under the stretched condition. In this chapter, we demonstrate various molecular complexes in SLI, and especially focus on membrane skeleton, protein 4.1G-membrane protein palmitoylated 6 (MPP6)-cell adhesion molecule 4 (CADM4). 4.1G was essential for the molecular targeting of MPP6 and CADM4 in SLI. Motor activity and myelin ultrastructures were abnormal in 4.1G-deficient mice, indicating the 4.1G function as a signal for proper formation of myelin in PNS. Thus, SLI probably has potential roles in the regulation of adhesion and signal transduction as well as in structural stability in Schwann cell myelin formation.

Immunohistochemical study of the membrane skeletal protein, membrane protein palmitoylated 6 (MPP6), in the mouse small intestine.

The membrane protein palmitoylated (MPP) family belongs to the membrane-associated guanylate kinase (MAGUK) family. MPP1 interacts with the protein 4.1 family member, 4.1R, as a membrane skeletal protein complex in erythrocytes. We previously described the interaction of another MPP family, MPP6, with 4.1G in the mouse peripheral nervous system. In the present study, the immunolocalization of MPP6 in the mouse small intestine was examined and compared with that of E-cadherin, zonula occludens (ZO)-1, and 4.1B, which we previously investigated in intestinal epithelial cells. The immunolocalization of MPP6 was also assessed in the small intestines of 4.1B-deficient (-/-) mice. In the small intestine, Western blotting revealed that the molecular weight of MPP6 was approximately 55-kDa, and MPP6 was immunostained under the cell membranes in the basolateral portions of almost all epithelial cells from the crypts to the villi. The immunostaining pattern of MPP6 in epithelial cells was similar to that of E-cadherin, but differed from that of ZO-1. In intestinal epithelial cells, the immunostained area of MPP6 was slightly different from that of 4.1B, which was restricted to the intestinal villi. The immunolocalization of MPP6 in small intestinal epithelial cells was similar between 4.1B(-/-) mice and 4.1B(+/+) mice. In the immunoprecipitation study, another MAGUK family protein, calcium/calmodulin-dependent serine protein kinase (CASK), was shown to molecularly interact with MPP6. Thus, we herein showed the immunolocalization and interaction proteins of MPP6 in the mouse small intestine, and also that 4.1B in epithelial cells was not essential for the sorting of MPP6.

Involvement of membrane skeletal molecules in the Schmidt-Lanterman incisure in Schwann cells.

Membrane skeletal networks form a two-dimensional lattice structure beneath erythrocyte membranes. 4.1R-MPP (membrane palmitoylated protein) 1-glycophorin C is one of the basic molecular complexes of the membrane skeleton. An analogous molecular complex, 4.1G-MPP6-cell adhesion molecule 4 (CADM4), is incorporated into the Schmidt-Lanterman incisure (SLI), a truncated cone shape in the myelin internode that is a specific feature of myelinated nerve fibers formed in Schwann cells in the peripheral nervous system. In this review, the dynamic structure of peripheral nerve fibers under stretching conditions is demonstrated using in vivo cryotechnique. The structures of nerve fibers had a beaded appearance, and the heights of SLI circular-truncated cones increased at the narrow sites of nerve fibers under the stretched condition. The height of SLI-truncated cones was lower in 4.1G-deficient nerve fibers than in wild-type nerve fibers. 4.1G was essential for the molecular targeting of MPP6 and CADM4 in SLI. The signal transduction protein, Src, was also involved in the 4.1G-MPP6-CADM4 molecular complex. The phosphorylation of Src was altered by the deletion of 4.1G. Thus, we herein demonstrate a membrane skeletal molecular complex in SLI that has potential roles in the regulation of adhesion and signal transduction as well as in structural stability in Schwann cells.

Histopathological changes in the periphery of the sciatic nerve of rats after knee joint immobilization.

[Purpose] This study was performed to investigate the histological changes that occur in the periphery of the sciatic nerve in rats undergoing knee immobilization. [Subjects and Methods] 29 male 9-week-old Wistar rats were divided randomly into a control group (C group, n = 7) and an immobilized group (I group, n = 22). The animals in the I group had the left knee joint immobilized in maximal flexion with plaster casts for two weeks. After the experimental period, we obtained cross-sections of tissues from the center of the left thigh, and the periphery of the sciatic nerve was observed under an optical microscope after hematoxylin-eosin staining. [Results] In contrast to the rats of C group, the rats in I group showed adherence between the bundle of nerve fibers and perineurium, as well as thickening of the perineurium. These histological changes were statistically significant. [Conclusions] Immobilization of the knee joints of rats resulted in characteristic histological changes in the connective tissue around the sciatic nerve.

Postural control exercise without using the upper limbs improves activities of daily living in patients with stroke.

Introduction: Stroke is one of the most common neurological disorders worldwide. Stroke survivors have restricted activities of daily living (ADL) and lower functional independence measures (FIM) after disease onset. Recovery of postural control abilities in patients with stroke is one of the most important therapeutic goals. In this study, we examined the differences in the FIM motor items between groups that performed postural control exercises with the upper limb and those that performed postural control exercises without the upper limb. Methods: The medical records of patients with stroke admitted and discharged from the Recovery Rehabilitation Unit at Azumino Red Cross Hospital between 2016 and 2018 were reviewed. We retrospectively investigated the relationships between postural control exercises with or without upper limbs, FIM motor items at admission and discharge, and percentage of gait acquisition at discharge. Results and Discussion: Among the thirteen FIM motor items, nine (bathing, dressing the upper body, dressing the lower body, toileting, transfers [bed, chair, and wheelchair], transfers [toilet], transfers [tub or shower], locomotion, and climbing of stairs) were significantly different between the two groups (those who performed postural control exercises with the upper limb and those who performed postural control exercises without the upper limb). Patients with stroke who performed postural control exercises without the upper limbs showed a higher percentage of gait acquisition. Touch contact during quiet standing reduces body sway and the associated fluctuations. However, continual practice of postural control with a small degree of body sway for a long period after a stroke would result in decreased pressure on the sole. This may hinder postural control relearning. Touch contact also reduces anticipatory postural adjustment, which may limit the improvement in balance ability during physical exercise. Postural control exercises without the upper limbs improve postural control ability and may be beneficial from a long-term perspective.

Recent Progress on Genetically Modified Animal Models for Membrane Skeletal Proteins: The 4.1 and MPP Families.

The protein 4.1 and membrane palmitoylated protein (MPP) families were originally found as components in the erythrocyte membrane skeletal protein complex, which helps maintain the stability of erythrocyte membranes by linking intramembranous proteins and meshwork structures composed of actin and spectrin under the membranes. Recently, it has been recognized that cells and tissues ubiquitously use this membrane skeletal system. Various intramembranous proteins, including adhesion molecules, ion channels, and receptors, have been shown to interact with the 4.1 and MPP families, regulating cellular and tissue dynamics by binding to intracellular signal transduction proteins. In this review, we focus on our previous studies regarding genetically modified animal models, especially on 4.1G, MPP6, and MPP2, to describe their functional roles in the peripheral nervous system, the central nervous system, the testis, and bone formation. As the membrane skeletal proteins are located at sites that receive signals from outside the cell and transduce signals inside the cell, it is necessary to elucidate their molecular interrelationships, which may broaden the understanding of cell and tissue functions.

A comprehensive analysis of multi-circulatory disorders in early pressure injury and their diagnostic significance in rat models.

Early pressure injury (PI) progression is associated with multi-circulatory disorders and they interplay with each other, resulting in a lack of a satisfactory diagnostic method. We generated early PI and blanchable erythema hairless rat models. Transparent disc method and capillary refilling time test (CRTT) results were recorded with ultraviolet camera to capture the dynamics changes, and the blanching index and refilling index were set for comprehensive analysis. The deteriorated areas of early PI showed non-blanchable erythema (NBE) and an increase in erythema at 0.5 and 6 h with the transparent disc method. CRTT showed a marked refilling delay at 12 h. The comprehensive analysis of blanching index and refilling index showed a significant change in erythema from NBE at 0.5 h and ischemia progressing to hemorrhage at 18 h. There was also a marked difference in the deteriorating and improving areas within the same erythema. Pathological analysis showed inflammatory cell infiltration, with marked edema accompanied by increased hemorrhage and tissue necrosis. Furthermore, small arteries and veins with thrombosis and microthrombi were observed. Consistent ischemia after decompression and subsequent hemorrhage are important indicators, and comprehensive analysis can help increase the positive diagnosis rate over that for other circulatory disorders alone.

Establishing an Appropriate Pressure for the Transparent Disc Method to Distinguish Early Pressure Injury and Blanchable Erythema.

Background: Non-blanchable erythema is used as a diagnostic indicator for stage 1 pressure injury (early PI); it is distinguished from blanchable erythema (BE) by the application of "light pressing". Considering the low of the accuracy of the degree of pressure applied, it is difficult to use this method in clinical settings. Methods: We constructed models of BE and early PI in order to determine the most appropriate pressure values using the transparent disc method. We observed erythema by using a Dermo-camera to quantify the gray and a* values of the wound area along with a spectrophotometer. Results: BE started to fade at 50 mmHg, while the gray values became statistically significant when the pressure was increased to 100 mmHg (p < 0.05). However, erythema remained even when the pressure was increased to 150 mmHg soon after decompression. By contrast, the early PI was showed to be non-blanchable for the longest time under a pressure of 150 mmHg, but by 18 h it had decreased and the erythema faded more obviously after applying pressure. Conclusions: We proposed that a pressure of 50−100 mmHg was more appropriate for light pressure, but this may vary when different instruments are used. Variations may occur in either BE or early PI, therefore, careful attention should be paid during observations.

Elucidation of Ischemic Mechanisms of Early Pressure Injury during Post-Decompression and Detecting Methods.

Background: Pressure injuries (PIs) generally result from prolonged ischemia through localized skin compression, and ischemia persists and exacerbates damage even post-decompression. The mechanisms of ischemia post-decompression are still unclear, and appropriate methods for detection are lacking. Methods: We used blanchable erythema (BE) and early PI rat models. We assessed the perfusion using Evans Blue (EB) and thrombus formation under a light microscope. Furthermore, we performed a capillary refill time test (CRTT) to detect ischemia after depression coupled with the transparent disk method using a spectrophotometer. Results: Compared with the BE group, the early PI group showed significantly slow and insufficient perfusion, as determined by EB staining (p < 0.001). Histological observations revealed that ischemia during post-decompression of early PI was caused by a greater amount of thrombi. The CRTT results showed that although both groups exhibited varying degrees of insufficient refilling volume, the early PI group had significantly slower refilling than the BE group (p < 0.001), which persisted during the deterioration or disappearance of erythema. Conclusions: Our results showed that persistent ischemia caused by thrombi is an important cause of early PI deterioration post-decompression. Therefore, the performance of CRTT coupled with the transparent disc method may become a promising method for detecting ischemia post-decompression.

Electron microscopic observation of photoreceptor cells in directly inserted anesthetized Drosophila into a high-pressure freezing unit.

The high-pressure freezing (HPF) technique is known to cryofix water-containing materials with little ice-crystal formation in deep depths compared with other freezing techniques. In this study, HPF for anesthetized living Drosophila was performed by placing them directly on the carrier of the HPF unit and exposing them to light. Frozen Drosophila were freeze substituted, and their compound eyes were examined by transmission electron microscopy. The ultrastructures of ommatidia composed of photoreceptor cells were well preserved. The location of the cytoplasmic organelles inside the photoreceptor cells was observed. In some photoreceptor cells in ommatidia of the light-exposed Drosphila, the cytoplasmic small granules were localized nearer the base of rhabdomeres, compared with those of the nonlight-exposed Drosophila. Thus, HPF with the direct insertion of living Drosophila under light exposure into the HPF machine enabled us to examine changes to functional structures of photoreceptor cells that occur within seconds.

Immunohistochemical study of mouse sciatic nerves under various stretching conditions with "in vivo cryotechnique".

BACKGROUND: In living animal bodies, some morphological changes of nerve fibers will probably occur when peripheral nerves are stretched or not stretched during various joint exercises. We aimed to capture the dynamic structures of nerves under various stretching conditions and to keep soluble serum proteins in their tissue sections. NEW METHOD: Morphological changes of stretched or non-stretched sciatic nerve fibers were examined with "in vivo cryotechnique" (IVCT). Fibers were directly frozen with liquid isopentane-propane cryogen (-193°C). Immunolocalizations of protein 4.1G and albumin were also examined in the fibers. RESULTS: The structures of IVCT-prepared sciatic nerves under the stretched condition showed a beaded appearance. By immunostaining for membrane skeletal protein 4.1G, Schmidt-Lanterman incisures (SLIs) were clearly identified, and the heights of their circular truncated cones were increased at narrow sites of the nerve fibers under the stretched condition, compared to those of non-stretched nerve fibers. Albumin was immunolocalized in blood vessels and also along endoneurium including regions near the node of Ranvier. COMPARISON WITH EXISTING METHODS: With the conventional perfusion-fixation method (PF), it was difficult to keep stable postures of living mouse limbs for tissue preparation. In nerve fibers after PF, the structures of SLI were easily modified, and albumin was heterogeneously immunolocalized due to diffusion artifacts. CONCLUSIONS: IVCT revealed (1) the structures of peripheral nerve fibers under dynamically different conditions, indicating that the morphological changes of SLIs play a functional role as a bumper structure against mechanical forces, and (2) accurate immunolocalization of serum albumin in the sciatic nerve fibers.