Blood-Nerve Barrier (BNB) Pathology in Diabetic Peripheral Neuropathy and In Vitro Human BNB Model.

In diabetic peripheral neuropathy (DPN), metabolic disorder by hyperglycemia progresses in peripheral nerves. In addition to the direct damage to peripheral neural axons, the homeostatic mechanism of peripheral nerves is disrupted by dysfunction of the blood-nerve barrier (BNB) and Schwann cells. The disruption of the BNB, which is a crucial factor in DPN development and exacerbation, causes axonal degeneration via various pathways. Although many reports revealed that hyperglycemia and other important factors, such as dyslipidemia-induced dysfunction of Schwann cells, contributed to DPN, the molecular mechanisms underlying BNB disruption have not been sufficiently elucidated, mainly because of the lack of in vitro studies owing to difficulties in establishing human cell lines from vascular endothelial cells and pericytes that form the BNB. We have developed, for the first time, temperature-sensitive immortalized cell lines of vascular endothelial cells and pericytes originating from the BNB of human sciatic nerves, and we have elucidated the disruption to the BNB mainly in response to advanced glycation end products in DPN. Recently, we succeeded in developing an in vitro BNB model to reflect the anatomical characteristics of the BNB using cell sheet engineering, and we established immortalized cell lines originating from the human BNB. In this article, we review the pathologic evidence of the pathology of DPN in terms of BNB disruption, and we introduce the current in vitro BNB models.

Local blood flow in peripheral nerves and their ganglia: Resurrecting key ideas around its measurement and significance.

Over 3 decades ago, seminal work by Phillip Low and colleagues established exquisite physiology around the measurement of nerve blood flow (NBF). Although not widely explored recently, its connection to the clinic has awaited human methodology. While human studies have not achieved a convincing level of rigour, newer imaging technologies are offering early information. The peripheral nerve trunk has parallel blood flow compartments that include epineurial flow dominated by arteriovenous shunts and downstream endoneurial blood flow (EBF). NBF and EBF have lower values than central nervous system blood flow, lack autoregulation yet have sympathetic and peptidergic neurovascular control. Contrary to expectation, injury to nerves is often associated with rises in NBF rather than ischemia, a finding of biological interest corroborated by human studies. Despite its potential importance, quantitative human measurements of EBF and NBF are not yet available. However, with development, careful NBF analysis may present new insights into nerve disorders. Muscle Nerve 57: 884-895, 2018.

Age-related morphological regression of myelinated fibers and capillary architecture of distal peripheral nerves in rats.

BACKGROUND: Regression of myelinated peripheral nerve fibers in the lower extremities contributes to sarcopenia and balance dysfunction in normal aging. This subclinical regression of myelinated fibers (MFs) is heavily influenced by alterations in microvasculature, though the mechanism underlying these age-related degenerative phenomena remains unclear. The aim of the present study was to examine age-related regressions in myelinated distal peripheral nerve fibers as well as capillary architecture in rats using both morphological and histochemical methods. RESULTS: MFs were categorized into tertiles of 'large', 'medium', and 'small' sizes based on the distribution of MF diameters. A two-way ANOVA was used to assess effects of fiber size (large/medium/small) and group (young/elderly) on myelin thickness, axon diameter, myelin perimeter, axon perimeter, and G-ratio (axon diameter/fiber diameter). Significant main effects were observed for both MF size and group with respect to all dimensions except for G-ratio. Values for fiber diameter (P < 0.01), myelin thickness (P < 0.01), axon diameter (P < 0.01), myelin perimeter (P < 0.01), and axon perimeter (P < 0.01) were significantly lower than those in the young group. Additionally, mean capillary diameter and number of microvascular branch points were significantly lower in the elderly group than in the young group. CONCLUSIONS: The present study demonstrated that spontaneous age-related regression predominantly occurs for all fiber sizes in the distal peripheral nerves and the capillary architecture. The results of the present study further suggest that both the distal MFs and capillaries in the peripheral nerve may simultaneously regress with aging.

The effects of capillary dysfunction on oxygen and glucose extraction in diabetic neuropathy.

Diabetic neuropathy is associated with disturbances in endoneurial metabolism and microvascular morphology, but the roles of these factors in the aetiopathogenesis of diabetic neuropathy remain unclear. Changes in endoneurial capillary morphology and vascular reactivity apparently predate the development of diabetic neuropathy in humans, and in manifest neuropathy, reductions in nerve conduction velocity correlate with the level of endoneurial hypoxia. The idea that microvascular changes cause diabetic neuropathy is contradicted, however, by reports of elevated endoneurial blood flow in early experimental diabetes, and of unaffected blood flow when early histological signs of neuropathy first develop in humans. We recently showed that disturbances in capillary flow patterns, so-called capillary dysfunction, can reduce the amount of oxygen and glucose that can be extracted by the tissue for a given blood flow. In fact, tissue blood flow must be adjusted to ensure sufficient oxygen extraction as capillary dysfunction becomes more severe, thereby changing the normal relationship between tissue oxygenation and blood flow. This review examines the evidence of capillary dysfunction in diabetic neuropathy, and whether the observed relation between endoneurial blood flow and nerve function is consistent with increasingly disturbed capillary flow patterns. The analysis suggests testable relations between capillary dysfunction, tissue hypoxia, aldose reductase activity, oxidative stress, tissue inflammation and glucose clearance from blood. We discuss the implications of these predictions in relation to the prevention and management of diabetic complications in type 1 and type 2 diabetes, and suggest ways of testing these hypotheses in experimental and clinical settings.

Vastus lateralis vascularized nerve graft in facial nerve reconstruction: an anatomical cadaveric study and clinical implications.

BACKGROUND: The present study investigates the vascular anatomy of the vastus lateralis motor nerve (VLMN) to be used as a vascularized nerve graft in facial nerve reconstruction. We evaluated the maximum length of the nerve that can be included in the flap and its vascular pedicle. In addition, we discuss its adequacy for use in early reconstruction of the facial nerve both as ipsilateral facial nerve reconstruction and as cross-facial nerve graft. METHODS: Five fresh cadavers were used in this study. In all specimens, the VLMN and its vascular pedicle were dissected, photodocumented and measured using calipers. In addition, two vascularized VLMN were injected with a radiopaque contrast and underwent CT angiography and three dimensional reconstructions were scanned to illustrate the vascular supply of the nerve using OsiriX Software. RESULTS: The VLMN was divided into two divisions, an oblique proximal and a descending distal, in 70% of the dissections with a mean maximal length of 8.4 ± 4.5 cm for the oblique division and 15.03 ± 3.87 cm for the descending division. The length of the oblique division, when present, was shorter than the length of the descending branch in all specimens. The mean length of the pedicle was 2.93 ± 1.69 cm, and 3.27 ± 1.49 cm until crossing the oblique and the descending division of the nerve respectively. The mean caliber of the nerve was 2.4 ± 0.62 mm. Three-dimensional computed tomography angiography demonstrated perfusion throughout the entire VLMN by branches from the descending branch of the lateral femoral circumflex artery which ran parallel to the descending division of the VLMN. Additionally, we observed that technically it was possible to preserve the oblique branch of the VLMN. CONCLUSION: This study confirms that VLMN presents adequate anatomic features to be used as a vascularized nerve graft for facial nerve reconstruction in terms of length, pedicle, and caliber.

Quantitative evaluation of blood perfusion to nerves included in the anterolateral thigh flap using indocyanine green fluorescence angiography: a different contrast pattern between the vastus lateralis motor nerve and femoral cutaneous nerve.

BACKGROUND: Better postoperative results can be expected in nerve reconstruction when vascularized nerve grafts are used. Previous studies reported reconstruction with flaps including "vascularized" nerves; however, few have evaluated blood supply to these nerves. The aim of this study was to quantitatively assess blood perfusion to nerves included in anterolateral thigh (ALT) flaps by indocyanine green (ICG) fluorescence angiography. PATIENTS AND METHODS: Participants comprised eight patients who underwent reconstructive surgery with nerve defects using free ALT flaps, including the vastus lateralis motor nerve and/or femoral cutaneous nerve. Intraoperative ICG fluorescent angiography was performed. Time after the drug injection and the intensity of fluorescence in these nerves were analyzed as time-intensity curves. Maximum intensity (Imax), time to Imax (Tmax), and time at the beginning of intensity elevation (Te) were measured at three points: Point C, the central portion of the flap-attached region of the nerve; Point P, 2 cm from the proximal flap-attached edge; and Point D, 2 cm from the distal edge. RESULTS: Imax and Te at point C and Imax/Tmax-Te at point P were significantly different between these two nerves (p = 0.03125, p = 0.02895, p = 0.03125, respectively). Fluorescence in the vastus lateralis motor nerve was slightly quicker and stronger than that in the femoral cutaneous nerve, and also exhibited an axial pattern of fluorescence. CONCLUSION: Intraoperative ICG fluorescent angiography can be used to determine which nerve is better for nerve reconstruction. The indexes of Imax, Te, and Imax/Tmax-Te may be the suitable criteria for decision making regarding donor nerve selection.

Investigation of venous drainage patterns of rabbit peripheral nerves.

In this study, the venous drainage patterns of white New Zealand rabbits' peripheral nerves were examined in an effort to provide an animal model for studies focusing on the venous drainage of the healing nerves. Extremities of rabbits sacrificed via intraarterial lead oxide-gelatin solution, and thus had their peripheral bloods pushed out of the arteries and into the veins were dissected for the venous drainage of radial, median, ulnar, femoral, sciatic, tibial and fibular nerves. The observations revealed that white New Zealand rabbit was a feasible model due to its consistent venous anatomy draining the major nerves and accessibility and workability of them. Of those dissected, the most suitable set of nerves suitable for such studies seem to be the Median nerve of the anterior extremity, and Sciatic nerve of the posterior extremity (Fig. 12, Ref. 16). Text in PDF www.elis.sk. Text in PDF www.elis.sk.

Painful and painless diabetic neuropathy: one disease or two?

Painful diabetic polyneuropathy (PDPN) is generally considered a variant of diabetic polyneuropathy (DPN) but the identification of distinctive aspects that characterize painful compared with painless DPN has however been addressed in many studies, mainly with the purpose of better understanding the mechanisms of neuropathic pain in the scenario of peripheral nerve damage of DPN, of determining risk markers for pain development, and also of recognizing who might respond to treatments. This review is aimed at examining available literature dealing with the issue of similarities and differences between painful and painless DPN in an attempt to respond to the question of whether painful and painless DPN are the same disease or not and to address the conundrum of why some people develop the insensate variety of DPN whilst others experience distressing pain. Thus, from the perspective of comparing painful with painless forms of DPN, this review considers the clinical correlates of PDPN, its distinctive framework of symptoms, signs, and nerve functional and structural abnormalities, the question of large and small fiber involvement, the peripheral pain mechanisms, the central processing of pain and some new insights into the pathogenesis of pain in peripheral polyneuropathies and PDPN.

Intrinsic microvasculature of the sciatic nerve in the rat.

Microvasculature associated with the sciatic nerve was examined using high-resolution micro-CT scanning in one group of rats and surgical exploration in another. The results indicate that blood supply to the sciatic nerve is an "open-ended" system in which the vessels run longitudinally within the epineurium and connect with external vasculature primarily at junction points. Although the range of vasculature found extended down to 4-5 µ, only a few isolated vessels of this size were found, with no capillary "mesh" as such, possibly because of the close proximity of the intrinsic vessel to nerve fibers within the epineurium. While the study did not include direct measurements of flow or nerve function, the "open-ended" pattern of vasculature found has important implications regarding the relationship between the two. Specifically, the nerve is less vulnerable to a severe or complete disruption in blood supply than it would be under a close-ended system such as that of the heart or brain, where a severe disruption can occur with the obstruction of only a single vessel. Indeed, the pattern of vasculature found, subject to further study of vasculature at the capillary level, suggests that flow within the intrinsic vessels may be in either direction, depending on circumstances, somewhat like flow within the circle of Willis in the cerebral circulation.

Punched nerve syndrome: ultrasonographic appearance of functional vascular nerve impairment.

PURPOSE: The mechanical impact of a neighboring vessel on a "punched" nerve segment is thought to be one possible cause of compression neuropathy but has not been proven definitively. We report on 9 subjects with unclear clinical mononeuropathies in whom we could clearly define peripheral nerve impairment by such vessels on real-time high-resolution ultrasound (HRUS). MATERIALS AND METHODS: Nine subjects with unclear mononeuropathy based on clinical neurological examination were referred to our department for HRUS assessment. The shape, inner and outer echotexture, size and diameter, and overall integrity of these nerves were assessed including an exact analysis of the surrounding soft tissues to search for potentially extraneural pathology. This included duplex imaging to identify even tiny atypical vascular structures. RESULTS: In all patients duplex HRUS showed the pulsatile and "punching" character of the relevant vessels and the direct mechanical impact of these vessel. The involved nerve segments appeared enlarged with a hypoechoic change of echotexture including at least partial masking of their inner fascicular texture. CONCLUSION: Although rare, a "punching" vessel can be the cause of a compression neuropathy. Therefore, duplex HRUS must be included in every HRUS examination of patients with otherwise unclear mononeuropathy.

Flow-Through Arterialized Posterior Interosseous Nerve Grafts for Digital Neurovascular Bundle Defects: Anatomical Study.

BACKGROUND: Digital neurovascular bundle defects are often encountered during crush or avulsion injuries and require complex reconstruction. Use of an arterialized nerve graft (neurovascular graft) serving both as an interpositional arterial conduit and as a nerve graft could be a reconstructive option in these cases. In this anatomical study, the authors aimed to describe a neurovascular graft of the posterior interosseous nerve and a branch of the anterior interosseous artery for neurovascular bundle reconstruction of the fingers. METHODS: Eighteen forearms were injected with red latex in order to collect the anatomical characteristics of the posterior interosseous nerve and the artery running near it. RESULTS: In all cases, the posterior interosseous nerve was followed by a branch of the anterior interosseous artery: the distal dorsal branch of the anterior interosseous nerve. The origin of this artery was proximal to the radiocarpal joint, at an average of 56.5 ± 11.1 mm. The proximal and distal diameters of the branch of the anterior interosseous artery were 1.6 ± 0.2 and 1.1 ± 0.2 mm, respectively. The proximal and distal diameters of the posterior interosseous nerve were 1.2 ± 0.3 mm and 1.1 ± 0.3 mm, respectively. CONCLUSIONS: These results show that a potential free neurovascular graft using the posterior interosseous nerve as nerve graft and the anterior interosseous artery as an arterial bypass to reconstruct both the nerve and arterial tree of the finger could be a useful approach. The authors speculate that this graft could be used to reconstruct the neurovascular bundle of amputated or devascularized digits.

Structure and barrier functions of the perineurium and its relationship with associated sensory corpuscles: A review.

Peripheral nerves are provided with a blood-nerve barrier which prevents the invasion of harmful substances and pathogens, and also regulates metabolic and ionic homeostasis within nerve fascicles. The barrier functions are attributed to both the concentric layer of flattened cells in the perineurium and blood vessels running in the endoneurium. The perineurial cells develop continuous tight junctions as a diffusion barrier. In order to take up a predominant nutrient, glucose, the perineurium as well as endoneurial capillaries expresses GLUT1, a glucose transporter. An axon-Schwann cell complex within peripheral nerves utilizes glucose as a major energy source via the GLUT1, as does the brain. Under conditions of a reduced utilization of glucose, only the perineurial cells can transfer other nutrients, namely monocarboxylates such as ketone bodies and lactate via MCT1. Thus, MCT1 colocalizes with GLUT1 in the perineurium but not in endoneurial capillaries. To identify the cellular origins of the nerve sheath, marker proteins such as glial specific S100 protein, GLUT1, endoneurial CD34, and EMA (epithelial membrane antigen) are useful. Immunohistochemical findings for these markers are reviewed in this paper, focusing on the perineurium and endoneurium and their derivatives, Pacinian and Meissner corpuscles. Growing evidence throws light on the critical involvement of the nerve sheaths in the development, maintenance, and diseases of peripheral nerves.

The effect of cigarette smoking on functional recovery following peripheral nerve ischemia/reperfusion injury.

This study was designed to determine if cigarette smoking adversely affects functional recovery following ischemia/reperfusion (I/R) injury in peripheral nerves. Forty Wistar rats were divided evenly among four groups. Animals in groups A and B were exposed to cigarette smoke via a controlled smoking chamber for 20 minutes daily. On study day 14, all animals underwent a controlled I/R injury to one sciatic nerve. Recovery was assessed with walking track assessments, malondialdehyde (MDA) assay, and histology. Walking track results on study day 21 did not differ significantly between the smoking and nonsmoking animals. However, by study day 28, the nonsmoking animals showed a greater degree of functional recovery (SFI = -18.0 and -22.8, respectively, P = 0.03). MDA concentration in the smoking group was significantly higher than the nonsmoking group at the 28 day time point (P = 0.04). Exposure to cigarette smoke was associated with a slower functional recovery following peripheral nerve I/R injury.

[Study of the vascular morphology of the lower limb nerves used as grafts]. / Estudio de la morfología vascular en los nervios de la extremidad inferior utilizados como injertos.

INTRODUCTION: The placement of a vascularized nervous graft is an option for acute nervous injuries treatment, as this has demonstrated a longer viability. OBJECTIVE: To carry out a macroscopic and microscopic analysis of the vascularity of the main lower limb nerves used as vascularized nervous grafts. MATERIALS AND METHODS: It was followed out a descriptive, transversal and non-comparative study to analyse the saphenous, the sural, the deep peroneal and the superficial peroneal nerves vascularity. This study was performed in two stages: 1) an in situ vascularity study and 2) a morphometric study. RESULTS: By the in situ study, it was demonstrated the following: the total length of the lower limb nerves used as graft, was in a decrease order: sural nerve (micro = 29.26 cm, +/- 4.05 cm), superficial peroneal nerve (micro = 28.61 cm, +/- 3.97 cm), deep peroneal nerve (micro = 26.64 cm, +/- 5.21 cm) and saphenous nerve (micro = 25.12 cm, +/- 4.42 cm). The morphometric study demonstrated that the dominant artery with the major diameter belongs to the saphenous nerve, with a diameter of 1.13 mm; then we have the superficial peroneal nerve with a 1.01 mm diameter, followed by the deep peroneal nerve with 0.91 mm, and finally the sural nerve with the smallest diameter, 0.85 mm. CONCLUSIONS: The saphenous nerve has the most appropriated morphological characteristics to be used as a vascularized graft; by the contrary the deep peroneal nerve represents the last option from all the nerves included in this study.

Effects of interleukin-6 treatment on neurovascular function, nerve perfusion and vascular endothelium in diabetic rats.

AIM: Interleukin-6 (IL-6), a member of the neuropoietic cytokine family, participates in neural development and has neurotrophic activity. Recent research has also indicated actions to improve vasa nervorum function in diabetes. Both these facets are potentially relevant for treatment of diabetic neuropathy. The aim of this study was to determine whether IL-6 treatment corrected changes in neurovascular function in streptozotocin-induced diabetic rats. METHODS: After 1 month of diabetes, rats were given IL-6 for 1 month. The rats were subjected to sensory testing and measurements of nerve conduction velocities and nerve blood flow by hydrogen clearance microelectrode polarography. Further groups were used to study responses of the isolated gastric fundus and renal artery. Results were statistically analysed using ANOVA and post hoc tests. RESULTS: Diabetic rats showed mechanical hyperalgesia, thermal hyperalgesia, and tactile allodynia. The former was unaffected by IL-6 treatment, whereas the latter two measures were corrected. Immunohistochemical staining of dorsal root ganglia for IL-6 did not reveal any changes with diabetes or treatment. The results showed that 22 and 17.4% slowing of sciatic motor and saphenous sensory nerve conduction velocities, respectively, with diabetes were improved by IL-6. Sciatic endoneurial perfusion was halved by diabetes and corrected by IL-6. A 40.6% diabetic deficit in maximal non-adrenergic, non-cholinergic relaxation of gastric fundus to nerve stimulation was unaffected by IL-6. Renal artery endothelium-dependent relaxation was halved by diabetes, the endothelium-derived hyperpolarizing factor (EDHF) component being severely attenuated. IL-6 did not affect nitric oxide-mediated vasorelaxation, but markedly improved EDHF responses. CONCLUSIONS: IL-6 improved aspects of small and large nerve fibre and vascular endothelium dysfunction in diabetic rats. The functional benefits related to increased nerve blood flow via an EDHF mechanism, and IL-6 could have therapeutic potential in diabetic neuropathy and vasculopathy, which should be further evaluated.

Is it possible to prefabricate a vascularized peripheral nerve graft?

The ideal technique to repair a damaged peripheral nerve is primary repair. Unfortunately, most damaged peripheral nerves have gaps making primary repair impossible. Autologous nerve grafts that are used to repair damaged nerves can either be conventional nonvascularized nerve grafts or vascularized nerve grafts. Vascularized nerve grafts are proposed to be superior to conventional nerve grafts especially in recipient beds that are scarred, with poor vascular supply. One of the disadvantages of vascularized nerve grafts is the limited donor site. It is possible to eliminate this problem by prefabricating vascularized nerve grafts. In this study, to prefabricate a vascularized nerve, segments harvested from left sciatic nerves of 10 Wistar albino rats were implanted on right femoral vessels, and intact right sciatic nerves were used as controls to evaluate the function, electrophysiologic studies, and histopathologic examination, were performed on these grafts 4 weeks after implantation. Prefabricated sciatic nerve grafts showed vascularization, but they did not show compound action potential activity to electrical stimulation and demonstrated diffuse and severe vacuolar degeneration and myelin loss. We were unable to prefabricate a functional vascularized nerve graft by this method.

[Anatomy of the artery of the cutaneous posterior nerve of the thigh]. / Anatomie de l'artère du nerf cutané postérieur de la cuisse.

Salmon has described first the vascularisation of the posterior cutaneous nerve in 1936. Since, few articles have described the collaterals of the artery accompanying the posterior cutaneous nerve. The authors conducted an anatomical study on 20 cadaveric dissections with injection in order to define the collaterals of the artery. The findings reveal an artery nourished proximally by fasciocutaneous branches of the profunda perforating arteries and distally by branches from popliteal and genicular arteries. Their association is variable.

A rapid, nondestructive method for vascular network visualization.

The quantitative analysis of blood vessel networks is an important component in many animal models of disease. We describe a nondestructive technique for blood vessel imaging that visualizes in situ vasculature in harvested tissues. The method allows for further analysis of the same tissues with histology and other methods that can be performed on fixed tissue. Consequently, it can easily be incorporated upstream to analysis methods to augment these with a three-dimensional reconstruction of the vascular network in the tissues to be analyzed. The method combines iodine-enhanced micro-computed tomography with a deep learning algorithm to segment vasculature within tissues. The procedure is relatively simple and can provide insight into complex changes in the vascular structure in the tissues.

The mouse olfactory peduncle 4: Development of synapses, perineuronal nets, and capillaries.

Olfaction is critical for survival in neonatal mammals. However, little is known about the neural substrate for this ability as few studies of synaptic development in several olfactory processing regions have been reported. Odor information detected in the nasal cavity is first processed by the olfactory bulb and then sent via the lateral olfactory tract to a series of olfactory cortical areas. The first of these, the anterior olfactory nucleus pars principalis (AONpP), is a simple, two layered cortex with an outer plexiform and inner cell zone (Layers 1 and 2, respectively). Five sets of studies examined age-related changes in the AONpP. First, immunocytochemistry for glutamatergic (VGlut1 and VGlut2) and GABAergic (VGAT) synapses demonstrated that overall synaptic patterns remained uniform with age. The second set quantified synaptic development with electron microscopy and found different developmental patterns between Layers 1 and 2. As many of the interhemispheric connections in the olfactory system arise from AONpP, the third set examined the development of crossed projections using anterograde tracers and electron microscopy to explore the maturation of this pathway. A fourth study examined ontogenetic changes in immunostaining for the proteoglycans aggrecan and brevican, markers of mesh-like extracellular structures known as perineuronal nets whose maturation is associated with the end of early critical periods of synaptogenesis. A final study found no age-related changes in the density of vasculature in the peduncle from P5 to P30. This work is among the first to examine early postnatal changes in this initial cortical region of the olfactory system.