Botulinum-toxin A (BoNT/A) is used for a wide range of conditions. Intramuscular administration of BoNT/A inhibits the release of acetylcholine at the neuromuscular junction from presynaptic motor neurons causing muscle-paralysis. The aim of the present study was to investigate the effect of high dose intramuscular BoNT/A injections (6 UI = 60 pg) on muscle tissue. The gait pattern of the rats was significantly affected 3 weeks after BoNT/A injection. The ankle joint rotated externally, the rats became flat footed, and the stride length decreased after BoNT/A injection. Additionally, there was clear evidence of microstructural changes on the tissue level by as evidenced by 3D imaging of the muscles by Synchrotron Radiation X-ray Tomographic Microscopy (SRXTM). Both the fibrillar and the non-fibrillar tissues were affected. The volume fraction of fibrillary tissue was reduced significantly and the non-fibrillar tissue increased. This was accompanied by a loss of the linear structure of the muscle tissue. Furthermore, gene expression analysis showed a significant upregulation of COL1A1, MMP-2, TGF-b1, IL-6, MHCIIA and MHCIIx in the BoNT/A injected leg, while MHVIIB was significantly downregulated. In conclusion: The present study reveals that high dose intramuscular BoNT/A injections cause microstructural damage of the muscle tissue, which contributes to impaired gait.

I.

Individuals with cerebral palsy (CP) develop skeletal muscle contractures that impair muscle function. In turn, contractures affect the ability to ambulate and often promote a sedentary lifestyle. The aim of the present study was to investigate the systemic inflammatory markers transforming growth factor beta-1 (TGFβ1), C-reactive protein (CRP), and interleukin-6 (IL-6) in children and adults with CP. Blood samples of n = 34 participants (24 individuals with CP (n = 14 children with CP age 10.36 ± 1.1 and n = 10 adults with CP age 38.80 ± 3.6) and 10 healthy adults age 36.63 ± 3.8) were analyzed for circulating levels of TGFβ1, CRP, and IL-6 using Sandwich Enzyme linked immunosorbent assay (ELISA) analyses (R&D systems). TGFβ1 and CRP levels were significantly higher in children with CP compared to both adults with CP (TGFβ1: P < 0.0005 and P < 0.0002, respectively) and healthy adults (CRP: P < 0.0001 and P < 0.0001, respectively), while no differences were observed between the adults with CP and healthy adults in TGFβ1 (P = 0.29) and CRP (P = 0.59), respectively. Furthermore, IL-6 levels showed no significant differences between the groups. The present findings indicate that the level of systemic inflammation is increased in children with CP. We speculate that persisting inflammation in children with CP might influence the development of muscle contractures, resulting in reduced muscle mass and marked muscle weakness in adults with CP.

The aim was to evaluate the performance of different microsampling materials in LC-MS-based protein analysis. The evaluated materials were the Volumetric Absorptive MicroSampling (VAMS) device, the pure cellulose sampling material (DMPK-C) and the water-soluble material (carboxymethyl cellulose, CMC), with the main emphasis on VAMS. Six proteins with different physicochemical properties were used as model proteins. A quick and generic sample preparation consisting of extraction/dissolution of the blood spot, tryptic digestion and subsequent matrix precipitation was applied prior to analysis. The recovery from VAMS, compared to DMPK-C, was dependent on the protein analyte: Lower recovery compared to DMPK-C was seen for β-lactoglobulin and myoglobin (74% and 80%, respectively) while higher recovery compared to DMPK-C was seen for cytochrome c and albumin (149% and 197%, respectively). The recovery from CMC was comparable to the recovery from DMPK-C for all proteins except for cytochrome c (76%). Hematocrit bias was evaluated for DMPK-C and VAMS, and the blood hematocrit influenced the protein analysis from both the materials. A preliminary evaluation was performed for VAMS: The correlation coefficient (R ≥ 0.983), the accuracy (71-101%) and the precision (RSD ≤ 20%) were determined for blood spiked with the six model proteins.

Muscle contractures are a common complication in patients with central nervous system (CNS) lesions which limit range of movement and cause joint deformities. Furthermore, it has previously been shown that muscles with contractures have a reduced number of capillaries, indicating decreased tissue vascularization. The aim of the present study was to investigate the microvascular volume (MV) at rest and after acute exercise in the muscle tissue of individuals with cerebral palsy (CP) and healthy control individuals. Contrast-enhanced ultrasound (CEUS) was used before and after 30 min of walking or running on a treadmill in 10 healthy control participants and 10 individuals with CP to detect MV of their skeletal muscle tissue. A significant increase in the MV was observed after exercise both in the adult CP group (21-53 yr) and in the control group (21-52 yr) (1.8 ± 0.8 ΔdB to 3.1 ± 0.9 ΔdB or 42.9% and 1.5 ± 0.6 ΔdB to 2.5 ± 0.9 ΔdB or 39.0%, respectively). Furthermore, a difference in the resting MV was observed between the most severe cases of CP [gross motor function classification scale (GMFCS) 3 and 4] (2.3 ± 0.5 ΔdB) and the less severe cases (GMFCS 1 and 2) (1.5 ± 0.2 ΔdB). When the CP group was walking (3.4 km/h), the lactate levels, Borg score, and heart rate matched the level of controls when they were running (9.8 km/h). In conclusion, individuals with CP become exhausted at much lower exercise intensities than healthy individuals. This is not explained by impaired microvascularization, since the MV of the individuals with CP respond normally to increased O demand during acute exercise. NEW & NOTEWORTHY Cerebral palsy (CP) patients were less physically active compared with typically developed individuals. This may affect the microvascularization. We observed that the CP group became exhausted at much lower exercise intensities compared with healthy individuals. However, impaired microvascularization was not the reason for the decreased physical activity as the CP group responded normally to increased O demand during acute exercise. These results indicate that walking may be recommended as an intervention to train and maintain skeletal muscle tissue in individuals with CP.