Functional recovery and muscle atrophy in pre-clinical models of peripheral nerve transection and gap-grafting in mice:effects of 4-aminopyridine

We recently demonstrated a repurposing beneficial effect of 4-aminopyridine(4-AP),a potassium channel blocker,on functional recove ry and muscle atrophy after sciatic nerve crush injury in rodents.However,this effect of 4-AP is unknown in nerve transection,gap,and grafting models.To evaluate and compare the functional recovery,nerve morphology,and muscle atrophy,we used a novel stepwise nerve transection with gluing(STG),as well as 7-mm irreparable nerve gap(G-7/0)and 7-mm isografting in 5-mm gap(G-5/7)models in the absence and presence of 4-AP treatment.Following surgery,sciatic functional index was determined wee kly to evaluate the direct in vivo global motor functional recovery.After 12 weeks,nerves were processed for whole-mount immunofluorescence imaging,and tibialis anterior muscles were harvested for wet weight and quantitative histomorphological analyses for muscle fiber crosssectional area and minimal Feret's diameter.Average post-injury sciatic functional index values in STG and G-5/7 models were significantly greater than those in the G-7/0 model.4-AP did not affect the sciatic functional index recovery in any model.Compared to STG,nerve imaging revealed more misdirected axons and distorted nerve architecture with isografting.While muscle weight,cross-sectional area,and minimal Feret's diameter were significantly smaller in G-7/0 model compared with STG and G-5/7,4-AP treatment significantly increased right TA muscle mass,cross-sectional area,and minimal Feret's diameter in G-7/0 model.These findings demonstrate that functional recovery and muscle atrophy after peripheral nerve injury are directly related to the intervening nerve gap,and 4-AP exerts diffe rential effects on functional recove ry and muscle atrophy.

Transcript shortening via alternative polyadenylation promotes gene expression during fracture healing

Maturation of the 3′end of almost all eukaryotic messenger RNAs(m RNAs)requires cleavage and polyadenylation.Most mammalian m RNAs are polyadenylated at different sites within the last exon,generating alternative polyadenylation(APA)isoforms that have the same coding region but distinct 3′untranslated regions(UTRs).The 3′UTR contains motifs that regulate m RNA metabolism;thus,changing the 3′UTR length via APA can significantly affect gene expression.Endochondral ossification is a central process in bone healing,but the impact of APA on gene expression during this process is unknown.Here,we report the widespread occurrence of APA,which impacts multiple pathways that are known to participate in bone healing.Importantly,the progression of endochondral ossification involves global 3′UTR shortening,which is coupled with an increased abundance of shortened transcripts relative to other transcripts;these results highlight the role of APA in promoting gene expression during endochondral bone formation.Our mechanistic studies of transcripts that undergo APA in the fracture callus revealed an intricate regulatory network in which APA enhances the expression of the collagen,type I,alpha 1(Col1a1)and Col1a2 genes,which encode the 2 subunits of the abundantly expressed protein collagen 1.APA exerts this effect by shortening the 3′UTRs of the Col1a1 and Col1a2 m RNAs,thus removing the binding sites of mi R-29a-3p,which would otherwise strongly promote the degradation of both transcripts.Taken together,our study is the first to characterize the crucial roles of APA in regulating the 3′UTR landscape and modulating gene expression during fracture healing.

Treatment of acute periprosthetic infections with prosthesis retention: Review of current concepts

Periprosthetic joint infection(PJI) is a devastating complication after total joint arthroplasty, occurring in approximately 1%-2% of all cases. With growing populations and increasing age, PJI will have a growing effect on health care costs. Many risk factors have been identified that increase the risk of developing PJI, including obesity, immune system deficiencies, malignancy, previous surgery of the same joint and longer operating time. Acute PJI occurs either postoperatively(4 wk to 3 mo after initial arthroplasty, depending on the classification system), or via hematogenous spreading after a period in which the prosthesis had functioned properly. Diagnosis and the choice of treatment are the cornerstones to success. Although different definitions for PJI have been used in the past, most are more or less similar and include the presence of a sinus tract, blood infection values, synovial white blood cell count, signs of infection on histopathological analysis and one ormore positive culture results. Debridement, antibiotics and implant retention(DAIR) is the primary treatment for acute PJI, and should be performed as soon as possible after the development of symptoms. Success rates differ, but most studies report success rates of around 60%-80%. Whether single or multiple debridement procedures are more successful remains unclear. The use of local antibiotics in addition to the administration of systemic antibiotic agents is also subject to debate, and its pro's and con's should be carefully considered. Systemic treatment, based on culture results, is of importance for all PJI treatments. Additionally, rifampin should be given in Staphylococcal PJIs, unless all foreign material is removed. The most important factors contributing to treatment failure are longer duration of symptoms, a longer time after initial arthroplasty, the need for more debridement procedures, the retention of exchangeable components, and PJI caused by Staphylococcus(aureus or coagulase negative). If DAIR treatment is unsuccessful, the following treatment option should be based on the patient health status and his or her expectations. For the best functional outcome, one- or two-stage revision should be performed after DAIR failure. In conclusion, DAIR is the obvious choice for treatment of acute PJI, with good success rates in selected patients.

Transdermal delivery of 4-aminopyridine accelerates motor functional recovery and improves nerve morphology following sciatic nerve crush injury in mice

Oral 4-aminopyridine(4-AP)is clinically used for symptomatic relief in multiple sclerosis and we recently demonstrated that systemic 4-AP had previously unknown clinically-relevant effects after traumatic peripheral nerve injury including the promotion of re-myelination,improvement of nerve conductivity,and acceleration of functional recovery.We hypothesized that,instead of oral or injection administration,transdermal 4-AP(TD-4-AP)could also improve functional recovery after traumatic peripheral nerve injury.Mice with surgical traumatic peripheral nerve injury received TD-4AP or vehicle alone and were examined for skin permeability,pharmacokinetics,functional,electrophysiological,and nerve morphological properties.4-AP showed linear pharmacokinetics and the maximum plasma 4-AP concentrations were proportional to TD-4-AP dose.While a single dose of TD-4-AP administration demonstrated rapid transient improvement in motor function,chronic TD-4-AP treatment significantly improved motor function and nerve conduction and these effects were associated with fewer degenerating axons and thicker myelin sheaths than those from vehicle controls.These findings provide direct evidence for the potential transdermal applicability of 4-AP and demonstrate that 4-AP delivered through the skin can enhance in-vivo functional recovery and nerve conduction while decreasing axonal degeneration.The animal experiments were approved by the University Committee on Animal Research(UCAR)at the University of Rochester(UCAR-2009-019)on March 31,2017.

Sacroiliac joint stability: Finite element analysis of implant number, orientation, and superior implant length

AIM To analyze how various implants placement variables affect sacroiliac(SI) joint range of motion. METHODS An experimentally validated finite element model of the lumbar spine and pelvis was used to simulate a fusion of the SI joint using various placement configurations of triangular implants(iF use Implant System~?). Placement configurations were varied by changing implant orientation, superior implant length, and number of implants. The range of motion of the SI joint was calculated using a constant moment of 10 N-m with a follower load of 400 N. The changes in motion were compared between the treatment groups to assess how the different variables affected the overall motion of the SI joint. RESULTS Transarticular placement of 3 implants with superior implants that end in the middle of the sacrum resulted in the greatest reduction in range of motion(flexion/extension = 73%, lateral bending = 42%, axial rotation = 72%). The range of motions of the SI joints were reduced with use of transarticular orientation(9%-18%) when compared with an inline orientation. The use of a superior implant that ended mid-sacrum resulted in median reductions of(8%-14%) when compared with a superior implant that ended in the middle of the ala. Reducing the number of implants, resulted in increased SI joint range of motions for the 1 and 2 implant models of 29%-133% and 2%-39%, respectively,when compared with the 3 implant model.CONCLUSION Using a validated finite element model we demonstrated that placement of 3 implants across the SI joint using a transarticular orientation with superior implant reaching the sacral midline resulted in the most stable construct. Additional clinical studies may be required to confirm these results.

The Effect of Ligament Modeling Technique on Knee Joint Kinematics: A Finite Element Study

Finite element (FE) analysis has become an increasingly popular technique in the study of human joint biomechanics, as it allows for detailed analysis of the joint/tissue behavior under complex, clinically relevant loading conditions. A wide variety of modeling techniques have been utilized to model knee joint ligaments. However, the effect of a selected constitutive model to simulate the ligaments on knee kinematics remains unclear. The purpose of the current study was to determine the effect of two most common techniques utilized to model knee ligaments on joint kinematics under functional loading conditions. We hypothesized that anatomic representations of the knee ligaments with anisotropic hyperelastic properties will result in more realistic kinematics. A previously developed, extensively validated anatomic FE model of the knee developed from a healthy, young female athlete was used. FE models with 3D anatomic and simplified uniaxial representations of main knee ligaments were used to simulate four functional loading conditions. Model predictions of tibiofemoral joint kinematics were compared to experimental measures. Results demonstrated the ability of the anatomic representation of the knee ligaments (3D geometry along with anisotropic hyperelastic material) in more physiologic prediction of the human knee motion with strong correlation (r ≥ 0.9 for all comparisons) and minimum deviation (0.9° ≤ RMSE ≤ 2.29°) from experimental findings. In contrast, non-physiologic uniaxial elastic representation of the ligaments resulted in lower correlations (r ≤ 0.6 for all comparisons) and substantially higher deviation (2.6°≤ RMSE ≤ 4.2°) from experimental results. Findings of the current study support our hypothesis and highlight the critical role of soft tissue modeling technique on the resultant FE predicted joint kinematics.

Human equivalent dose of oral 4-aminopyridine differentiates nerve crush injury from transection injury and improves post-injury function in mice

4-Aminopyridine(4-AP), an FDA-approved drug for the symptomatic treatment of multiple sclerosis, is used to improve neuromuscular function in patients with diverse demyelinating disorders. We recently demonstrated that local, transdermal or injectable forms of 4-AP improve myelination, nerve conduction velocity, muscle atrophy, and motor function after traumatic peripheral nerve injury in mice. While oral 4-AP is most commonly used in the clinic, it is unknown whether human equivalent oral doses of 4-AP have effects on traumatic peripheral nerve injury differentiation, myelination, muscle atrophy, functional recovery, and post-injury inflammatory processes in animals. Mice with sciatic nerve crush or denervation injury received oral or intraperitoneal 4-AP(10 μg) or vehicle alone and were examined for pharmacokinetics, motor function, muscle mass, intrinsic muscle force, nerve morphological and gene expression profiles. 4-AP showed linear pharmacokinetics and the maximum plasma 4-AP concentrations were proportional to 4-AP dose. Acute single dose of oral 4-AP administration induced a rapid transient improvement in motor function that was different in traumatic peripheral nerve injury with or without nerve continuity, chronic daily oral 4-AP treatment significantly enhanced post crush injury motor function recovery and this effect was associated with improved myelination, muscle mass, and ex vivo muscle force. Polymerase chain reaction array analysis with crushed nerve revealed significant alterations in gene involved in axonal inflammation and regeneration. These findings provide convincing evidence that regardless of the route of administration, 4-AP can acutely differentiate traumatic peripheral nerve injury with or without nerve continuity and can enhance in vivo functional recovery with better preservation of myelin sheaths, muscle mass, and muscle force. The animal experiments were approved by the University Committee on Animal Research(UCAR) at the University of Rochester(UCAR-2009-019) on March 31, 2017.

The Endplate Morphology Changes with Change in Biomechanical Environment Following Discectomy

Bone is a dynamic structure and is known to respond to changes in the load over time, in accordance with Wolff’s law. It states that the bone changes its shape and internal architecture in response to stresses acting on it [1]. Therefore, any structural changes in the spine may lead to bone remodeling due to changes in the optimal stress pattern. The changes in apparent density and thickness of the endplates following discectomy of varying amounts were analyzed. The study design coupled a bone remodeling algorithm based on strain energy density theory of adaptive remodeling with an experimentally validated 3D ligamentous finite element model of the spine. The apparent density and thickness of the index level endplates decreased above and below the region of discectomy. On the other hand, these parameters showed increases at the remaining regions of the endplate. There were no correlations between the amount of nucleus removed and the average percentage changes in apparent density and thickness of endplate above and below the discectomy region. However, the average percentage changes in apparent density and thickness at endplate in the other region increased with increase in amount of nucleus removed. These predictions are in agreement with the clinical observations [2-6].

Normal age-related viscoelastic properties of chondrons and chondrocytes isolated from rabbit knee

Background The mechanical microenvironment of the chondrocytes plays an important role in cartilage homeostasis and in the health of the joint. The pericellular matrix, cellular membrane of the chondrocytes, and their cytoskeletal structures are key elements in the mechanical environment. The aims of this study are to measure the viscoelastic properties of isolated chondrons and chondrocytes from rabbit knee cartilage using micropipette aspiration and to determine the effect of aging on these properties. Methods Three age groups of rabbit knees were evaluated： （1） young （2 months, n=10）; （2） adult （8 months, n=10）; and （3） old （31 months, n=10）. Chondrocytes were isolated from the right knee cartilage and chondrons were isolated from left knees using enzymatic methods. Micropipette aspiration combined with a standard linear viscoelastic solid model was used to quantify changes in the viscoelastic properties of chondrons and chondrocytes within 2 hours of isolation. The morphology and structure of isolated chondrons were evaluated by optical microscope using hematoxylin and eosin staining and collagen-6 immunofluorescence staining. Results In response to an applied constant 0.3-0.4 kPa of negative pressure, all chondrocytes exhibited standard linear viscoelastic solid properties. Model predictions of the creep data showed that the average equilibrium modulus （E~）, instantaneous modulus （E0）, and apparent viscosity （~） of old chondrocytes was significantly lower than the young and adult chondrocytes （P 〈0.001）; however, no difference was found between young and adult chondrocytes （P 〉0.05）. The adult and old chondrons generally possessed a thicker pericellular matrix （PCM） with more enclosed cells. The young and adult chondrons exhibited the same viscoelastic creep behavior under a greater applied pressure （1.0-1.1 kPa） without the deformation seen in the old chondrons. The viscoelastic properties （E,, E0, and/~） of young and adult chondrons were significantly greater than that observed in young and adult cells, respectively （P 〈0.001）. The adult chondrons were stiffer than the young chondrons under micropipette aspiration （P 〈0.001）. Conclusions Our findings provide a theoretical model to measure the viscoelastic properties of the chondrons as a whole unit by micropipette aspiration, and further suggest that the properties of the chondrocytes and PCM have an important influence on the biomechanical microenvironment of the knee joint cartilage degeneration that occurs with aging.