Peripheral nerve regeneration following scaffold-free conduit transplant of autologous dermal fibroblasts: a non-randomised safety and feasibility trial.

BACKGROUND: The use of Bio 3D nerve conduits is a promising approach for peripheral nerve reconstruction. This study aimed to assess their safety in three patients with peripheral nerve defects in their hands. METHODS: We describe a single institution, non-blinded, non-randomised control trial conducted at Kyoto University Hospital. Eligibility criteria included severed peripheral nerve injuries or a defect in the region distal to the wrist joint not caused by a congenital anomaly; a defect with a length of ≤20 mm in a nerve with a diameter ≤2 mm; failed results of sensory functional tests; ability to register in the protocol within 6 months from the day of injury; refusal of artificial nerve or autologous nerve transplantation; age 20-60 years; and willingness to participate and provide informed written consent. Six weeks before transplantation, skin was harvested, dermal fibroblasts were isolated and expanded, and Bio 3D nerve conduits were created using a Bio 3D printer. Bio 3D nerve conduits were transplanted into the patients' nerve defects. The safety of Bio 3D nerve conduits in patients with a peripheral nerve injury in the distal part of the wrist joint were assessed over a 48-week period after transplantation. RESULTS: No adverse events related to the use of Bio 3D nerve conduits were observed in any patient, and all three patients completed the trial. CONCLUSIONS: Bio 3D nerve conduits were successfully used for clinical nerve reconstruction without adverse events and are a possible treatment option for peripheral nerve injuries.

Physical injuries often result in damage to nerves, for example, in the hands. Replacement of the nerve with nerves removed from elsewhere in the patient's body is often the suggested treatment when the nerve is unable to repair itself. As an alternative to remove healthy nerve from elsewhere in the body, we used an adapted printer to create an artificial nerve equivalent from skin cells obtained from the patient's skin. We reconstructed the nerves of three individual with nerve defects in their hands, and we found that the function of the nerve improved, and the people did not experience negative consequences. This approach could be used widely to repair damaged nerves.

Safety of Silk-elastin Sponges in Patients with Chronic Skin Ulcers: A Phase I/II, Single-center, Open-label, Single-arm Clinical Trial.

BACKGROUND: Although traditional wound dressings such as collagen scaffolds promote granulation tissue formation, the efficacy of these dressings in chronic wounds is limited because of high susceptibility to bacterial growth. Biomaterials that can be applied to chronic wounds should have an anti-bacterial function. We previously reported that administering a silk-elastin solution that forms moisturizing hydrogels to wound surfaces of diabetic mice reduced bacterial growth and promoted granulation tissue formation compared with control or carboxymethyl cellulose hydrogels. We hypothesized that silk-elastin promotes wound healing in human chronic wounds by suppressing bacterial growth. METHODS: An open-label, clinical case series was conducted with a prospective, single-arm design at Kyoto University Hospital in Kyoto, Japan. In this study, 6 patients with chronic skin ulcers of any origin (2 < ulcer area (cm2) < 25) on their lower extremities were included; patients with critical ischemia were excluded. Silk-elastin sponges were applied and covered with a polyurethane film without changing the dressing for 14 days. Inflammation triggered treatment discontinuation due to fear of infection. The primary study endpoint was adverse events, including inflammation and infection. RESULTS: Poor hydrogel formation, possibly due to continuous exudation, was observed. No serious adverse events were noted. Two patients discontinued treatment on day 6 and day 7, respectively, due to inflammation, but they were not infected. The other 4 patients completed the 14-day silk-elastin sponge treatment without infection. CONCLUSION: Silk-elastin sponge is safe for chronic skin ulcers, and its ability to promote wound healing should be determined by confirmatory clinical trials.

The Efficacy of a Scaffold-free Bio 3D Conduit Developed from Autologous Dermal Fibroblasts on Peripheral Nerve Regeneration in a Canine Ulnar Nerve Injury Model: A Preclinical Proof-of-Concept Study.

Autologous nerve grafting is widely accepted as the gold standard treatment for segmental nerve defects. To overcome the inevitable disadvantages of the original method, alternative methods such as the tubulization technique have been developed. Several studies have investigated the characteristics of an ideal nerve conduit in terms of supportive cells, scaffolds, growth factors, and vascularity. Previously, we confirmed that biological scaffold-free conduits fabricated from human dermal fibroblasts promote nerve regeneration in a rat sciatic nerve injury model. The purpose of this study is to evaluate the feasibility of biological scaffold-free conduits composed of autologous dermal fibroblasts using a large-animal model. Six male beagle dogs were used in this study. Eight weeks before surgery, dermal fibroblasts were harvested from their groin skin and grown in culture. Bio 3D conduits were assembled from proliferating dermal fibroblasts using a Bio 3D printer. The ulnar nerve in each dog's forelimb was exposed under general anesthesia and sharply cut to create a 5 mm interstump gap, which was bridged by the prepared 8 mm Bio 3D conduit. Ten weeks after surgery, nerve regeneration was investigated. Electrophysiological studies detected compound muscle action potentials (CMAPs) of the hypothenar muscles and motor nerve conduction velocity (MNCV) in all animals. Macroscopic observation showed regenerated ulnar nerves. Low-level hypothenar muscle atrophy was confirmed. Immunohistochemical, histological, and morphometric studies confirmed the existence of many myelinated axons through the Bio 3D conduit. No severe adverse event was reported. Hypothenar muscles were re-innervated by regenerated nerve fibers through the Bio 3D conduit. The scaffold-free Bio 3D conduit fabricated from autologous dermal fibroblasts is effective for nerve regeneration in a canine ulnar nerve injury model. This technology was feasible as a treatment for peripheral nerve injury and segmental nerve defects in a preclinical setting.

Control of excitatory hierarchical circuits by parvalbumin-FS basket cells in layer 5 of the frontal cortex: insights for cortical oscillations.

The cortex contains multiple neuron types with specific connectivity and functions. Recent progress has provided a better understanding of the interactions of these neuron types as well as their output organization particularly for the frontal cortex, with implications for the circuit mechanisms underlying cortical oscillations that have cognitive functions. Layer 5 pyramidal cells (PCs) in the frontal cortex comprise two major subtypes: crossed-corticostriatal (CCS) and corticopontine (CPn) cells. Functionally, CCS and CPn cells exhibit similar phase-dependent firing during gamma waves but participate in two distinct subnetworks that are linked unidirectionally from CCS to CPn cells. GABAergic parvalbumin-expressing fast-spiking (PV-FS) cells, necessary for gamma oscillation, innervate PCs, with stronger and global inhibition to somata and weaker and localized inhibitions to dendritic shafts/spines. While PV-FS cells form reciprocal connections with both CCS and CPn cells, the excitation from CPn to PV-FS cells exhibits short-term synaptic dynamics conducive for oscillation induction. The electrical coupling between PV-FS cells facilitates spike synchronization among PV-FS cells receiving common excitatory inputs from local PCs and inhibits other PV-FS cells via electrically communicated spike afterhyperpolarizations. These connectivity characteristics can promote synchronous firing in the local networks of CPn cells and firing of some CCS cells by anode-break excitation. Thus subsets of L5 CCS and CPn cells within different levels of connection hierarchy exhibit coordinated activity via their common connections with PV-FS cells, and the resulting PC output drives diverse neuronal targets in cortical layer 1 and the striatum with specific temporal precision, expanding the computational power of the cortical network.

Temporal Structure of Neuronal Activity among Cortical Neuron Subtypes during Slow Oscillations in Anesthetized Rats.

Slow-wave oscillations, the predominant brain rhythm during sleep, are composed of Up/Down cycles. Depolarizing Up-states involve activity in layer 5 (L5) of the neocortex, but it is unknown how diverse subtypes of neurons within L5 participate in generating and maintaining Up-states. Here we compare the in vivo firing patterns of corticopontine (CPn) pyramidal cells, crossed-corticostriatal (CCS) pyramidal cells, and fast-spiking (FS) GABAergic neurons in the rat frontal cortex, with those of thalamocortical neurons during Up/Down cycles in the anesthetized condition. During the transition from Down- to Up-states, increased activity in these neurons was highly temporally structured, with spiking occurring first in thalamocortical neurons, followed by cortical FS cells, CCS cells, and, finally, CPn cells. Activity in some FS, CCS, and CPn neurons occurred in phase with Up-nested gamma rhythms, with FS neurons showing phase delay relative to pyramidal neurons. These results suggest that thalamic and cortical pyramidal neurons are activated in a specific temporal sequence during Up/Down cycles, but cortical pyramidal cells are activated at a similar gamma phase. In addition to Up-state firing specificity, CCS and CPn cells exhibited differences in activity during cortical desynchronization, further indicating projection- and state-dependent information processing within L5. SIGNIFICANCE STATEMENT: Patterned activity in neocortical electroencephalograms, including slow waves and gamma oscillations, is thought to reflect the organized activity of neocortical neurons that comprises many specialized neuron subtypes. We found that the timing of action potentials during slow waves in individual cortical neurons was correlated with their laminar positions and axonal targets. Within gamma cycles nested in the slow-wave depolarization, cortical pyramidal cells fired earlier than did interneurons. At the start of slow-wave depolarizations, activity in thalamic neurons receiving inhibition from the basal ganglia occurred earlier than activity in cortical neurons. Together, these findings reveal a temporally ordered pattern of output from diverse neuron subtypes in the frontal cortex and related thalamic nuclei during neocortical oscillations.

Multiple layer 5 pyramidal cell subtypes relay cortical feedback from secondary to primary motor areas in rats.

Higher-order motor cortices, such as the secondary motor area (M2) in rodents, select future action patterns and transmit them to the primary motor cortex (M1). To better understand motor processing, we characterized "top-down" and "bottom-up" connectivities between M1 and M2 in the rat cortex. Somata of pyramidal cells (PCs) in M2 projecting to M1 were distributed in lower layer 2/3 (L2/3) and upper layer 5 (L5), whereas PCs projecting from M1 to M2 had somata distributed throughout L2/3 and L5. M2 afferents terminated preferentially in upper layer 1 of M1, which also receives indirect basal ganglia output through afferents from the ventral anterior and ventromedial thalamic nuclei. On the other hand, M1 afferents terminated preferentially in L2/3 of M2, a zone receiving indirect cerebellar output through thalamic afferents from the ventrolateral nucleus. While L5 corticopontine (CPn) cells with collaterals to the spinal cord did not participate in corticocortical projections, CPn cells with collaterals to the thalamus contributed preferentially to connections from M2 to M1. L5 callosal projection (commissural) cells participated in connectivity between M1 and M2 bidirectionally. We conclude that the connectivity between M1 and M2 is directionally specialized, involving specific PC subtypes that selectively target lamina receiving distinct thalamocortical inputs.

Differentiated participation of thalamocortical subnetworks in slow/spindle waves and desynchronization.

During sleep, the electroencephalogram exhibits synchronized slow waves that desynchronize when animals awaken [desynchronized states (DSs)]. During slow-wave states, the membrane potentials of cortical neurons oscillate between discrete depolarized states ("Up states") and periods of hyperpolarization ("Down states"). To determine the role of corticothalamic loops in generating Up/Down oscillations in rats, we recorded unit activities of layer 5 (L5) corticothalamic (CTh) cells in the frontal cortex, neurons in the thalamic reticular nucleus, and basal ganglia- and cerebellum-linked thalamic relay nuclei, while simultaneously monitoring the local cortical field potential to identify slow-wave/spindle oscillations and desynchronization. We found that (1) some basal ganglia-linked and reticular thalamic cells fire preferentially near the beginning of Up states; (2) thalamic cells fire more selectively at a given Up-state phase than do CTh cells; (3) CTh and thalamic cells exhibit different action potential timings within spindle cycles; and (4) neurons exhibit different firing characteristics when comparing their activity during Up states and DSs. These data demonstrate that cortico-thalamo-cortical subnetworks are temporally differentiated during slow and spindle oscillations, that the basal ganglia-linked thalamic nuclei are closely related with Up-state initiation, and that Up states and DSs are distinguished as different depolarization states of neurons within the network.

Serotonin modulates fast-spiking interneuron and synchronous activity in the rat prefrontal cortex through 5-HT1A and 5-HT2A receptors.

Alterations of the serotonergic system in the prefrontal cortex (PFC) are implicated in psychiatric disorders such as schizophrenia and depression. Although abnormal synchronous activity is observed in the PFC of these patients, little is known about the role of serotonin (5-HT) in cortical synchrony. We found that 5-HT, released by electrical stimulation of the dorsal raphe nucleus (DRN) in anesthetized rats, regulates the frequency and the amplitude of slow (<2 Hz) waves in the PFC via 5-HT(2A) receptors (5-HT(2A)Rs). 5-HT also modulates prefrontal gamma (30-80 Hz) rhythms through both 5-HT(1A)Rs and 5-HT(2A)Rs, but not 5-HT(2C)Rs, inducing an overall decrease in the amplitude of gamma oscillations. Because fast-spiking interneurons (FSi) are involved in the generation of gamma waves, we examined serotonergic modulation of FSi activity in vivo. Most FSi are inhibited by serotonin through 5-HT(1A)Rs, while a minority is activated by 5-HT(2A)Rs, and not 5-HT(2C)Rs. In situ hybridization histochemistry confirmed that distinct populations of FSi in the PFC express 5-HT(1A)Rs and 5-HT(2A)Rs, and that the number of FSi expressing 5-HT(2C)Rs is negligible. We conclude that 5-HT exerts a potent control on slow and gamma oscillations in the PFC. On the one hand, it shapes the frequency and amplitude of slow waves through 5-HT(2A)Rs. On the other hand, it finely tunes the amplitude of gamma oscillations through 5-HT(2A)R- and 5-HT(1A)R-expressing FSi, although it primarily downregulates gamma waves via the latter population. These results may provide insight into impaired serotonergic control of network activity in psychiatric illnesses such as schizophrenia and depression.

Two distinct activity patterns of fast-spiking interneurons during neocortical UP states.

During sleep, neocortical neuronal networks oscillate slowly (<1 Hz) between periods of activity (UP states) and silence (DOWN states). UP states favor the interaction between thalamic-generated spindles (7-14 Hz) and cortically generated gamma (30-80 Hz) waves. We studied how these three nested oscillations modulate fast-spiking interneuron (FSi) activity in vivo in VGAT-Venus transgenic rats. Our data describe a population of FSi that discharge "early" within UP states and another population that discharge "late." Early FSi tended to be silent during epochs of desynchronization, whereas late FSi were active. We hypothesize that late FSi may be responsible for generating the gamma oscillations associated with cognitive processing during wakefulness. Remarkably, FSi populations were differently modulated by spindle and gamma rhythms. Early FSi were robustly coupled to spindles and always discharged earlier than late FSi within spindle and gamma cycles. The preferred firing phase during spindle and gamma waves was strongly correlated in each cell, suggesting a cross-frequency coupling between oscillations. Our results suggest a precise spatiotemporal pattern of FSi activity during UP states, whereby information rapidly flows between early and late cells, initially promoted by spindles and efficiently extended by local gamma oscillations.