Oxygen transport kinetics underpin rapid and robust diaphragm recovery following chronic spinal cord injury

Months after spinal cord injury (SCI), respiratory deficits remain the primary cause of morbidity and mortality for patients. It is possible to induce partial respiratory motor functional recovery in chronic SCI following 2 weeks of spinal neuroplasticity. However, the peripheral mechanisms underpinning this recovery are largely unknown, limiting development of new clinical treatments with potential for complete functional restoration. Utilizing a rat hemisection model, diaphragm function and paralysis was assessed and recovered at chronic time points following trauma through chondroitinase ABC induced neuroplasticity. We simulated the diaphragm's in vivo cyclical length change and activity patterns using the work loop technique at the same time as assessing global and local measures of the muscles histology to quantify changes in muscle phenotype, microvascular composition, and oxidative capacity following injury and recovery. These data were fed into a physiologically informed model of tissue oxygen transport. We demonstrate that hemidiaphragm paralysis causes muscle fibre hypertrophy, maintaining global oxygen supply, although it alters isolated muscle kinetics, limiting respiratory function. Treatment induced recovery of respiratory activity normalized these effects, increasing oxygen supply, restoring optimal diaphragm functional properties. However, metabolic demands of the diaphragm were significantly reduced following both injury and recovery, potentially limiting restoration of normal muscle performance. The mechanism of rapid respiratory muscle recovery following spinal trauma occurs through oxygen transport, metabolic demand and functional dynamics of striated muscle. Overall, these data support a systems‐wide approach to the treatment of SCI, and identify new targets to mediate complete respiratory recovery

Effects of treadmill training on microvascular remodeling in the rat following spinal cord injury

Introduction: The morphological characteristics of skeletal muscles innervated caudal to a spinal cord injury (SCI) undergo dramatic phenotypic and microvascular changes. Method: Female Sprague Dawley rats received a severe contusion at thoracic level 9/10, and were randomly assigned to locomotor training (TR), epidural stimulation (ES) or a combination of the treatment groups (CB). Fibre type composition and capillary distribution were assessed in phenotypically distinct compartments of the tibialis anterior. Results: SCI induced a shift in Type II fibre phenotype from oxidative to glycolytic (P<0.05) as well as capillary loss within the oxidative core and glycolytic cortex; the CB treatment best maintained capillary supply within both compartments. Discussion: The angiogenic response of CB training improved capillary distribution across the muscle, becoming spatially more homogeneous and decreasing mean capillary supply area, potentially improving oxygenation. There is an important role for weight bearing training in maintaining the oxidative phenotype of muscle following SCI

C‐bouton components on rat extensor digitorum longus motoneurons are resistant to chronic functional overload

Mammalian motor systems adapt to the demands of their environment. For example, muscle fibre types change in response to increased load or endurance demands. However, for adaptations to be effective, motoneurons must adapt such that their properties match those of the innervated muscle fibres. We used a rat model of chronic functional overload to assess adaptations to both motoneuron size and a key modulatory synapse responsible for amplification of motor output, C‐boutons. Overload of extensor digitorum longus (EDL) muscles was induced by removal of their synergists, tibialis anterior muscles. Following 21 days survival, EDL muscles showed an increase in fatigue resistance and a decrease in force output, indicating a shift to a slower phenotype. These changes were reflected by a decrease in motoneuron size. However, C‐bouton complexes remained largely unaffected by overload. The C‐boutons themselves, quantified by expression of vesicular acetylcholine transporter, were similar in size and density in the control and overload conditions. Expression of the post‐synaptic voltage‐gated potassium channel (KV2.1) was also unchanged. Small conductance calcium‐activated potassium channels (SK3) were expressed in most EDL motoneurons, despite this being an almost exclusively fast motor pool. Overload induced a decrease in the proportion of SK3+ cells, however, there was no change in density or size of clusters. We propose that reductions in motoneuron size may promote early recruitment of EDL motoneurons, but that C‐bouton plasticity is not necessary to increase the force output required in response to muscle overload

Abnormal skeletal muscle blood flow, contractile mechanics and fibre morphology in a rat model of obese‐HFpEF

Peripheral skeletal muscle and vascular alterations induced by heart failure with preserved ejection fraction (HFpEF) remain poorly identified, with limited therapeutic targets. This study used a cardiometabolic obese‐HFpEF rat model to comprehensively phenotype skeletal muscle mechanics, blood flow, microvasculature and fibre atrophy. Lean (n = 8) and obese‐HFpEF (n = 8) ZSF1 rats were compared. Skeletal muscles (soleus and diaphragm) were assessed for in vitro contractility (isometric and isotonic properties) alongside indices of fibre‐type cross‐sectional area, myosin isoform, and capillarity, and estimated muscle PO2. In situ extensor digitorum longus (EDL) contractility and femoral blood flow were assessed. HFpEF soleus demonstrated lower absolute maximal force by 22%, fibre atrophy by 24%, a fibre‐type shift from I to IIa, and a 17% lower capillary‐to‐fibre ratio despite increased capillary density (all P 0.05). Soleus isotonic properties (shortening velocity and power) were impaired by up to 17 and 22%, respectively (P < 0.05), while the magnitude of the exercise hyperaemia was attenuated by 73% (P = 0.012) in line with higher muscle fatigue by 26% (P = 0.079). Diaphragm alterations (P < 0.05) included Type IIx fibre atrophy despite Type I/IIa fibre hypertrophy, with increased indices of capillarity alongside preserved contractile properties during isometric, isotonic, and cyclical contractions. In conclusion, obese‐HFpEF rats demonstrated blunted skeletal muscle blood flow during contractions in parallel to microvascular structural remodelling, fibre atrophy, and isotonic contractile dysfunction in the locomotor muscles. In contrast, diaphragm phenotype remained well preserved. This study identifies numerous muscle‐specific impairments that could exacerbate exercise intolerance in obese‐HFpEF

Regional variation of the cortical and trabecular bone material properties in the rabbit skull

The material properties of some bones are known to vary with anatomical location, orientation and position within the bone (e.g., cortical and trabecular bone). Details of the heterogeneity and anisotropy of bone is an important consideration for biomechanical studies that apply techniques such as finite element analysis, as the outcomes will be influenced by the choice of material properties used. Datasets detailing the regional variation of material properties in the bones of the skull are sparse, leaving many finite element analyses of skulls no choice but to employ homogeneous, isotropic material properties, often using data from a different species to the one under investigation. Due to the growing significance of investigating the cranial biomechanics of the rabbit in basic science and clinical research, this study used nanoindentation to measure the elastic modulus of cortical and trabecular bone throughout the skull. The elastic moduli of cortical bone measured in the mediolateral and ventrodorsal direction were found to decrease posteriorly through the skull, while it was evenly distributed when measured in the anteroposterior direction. Furthermore, statistical tests showed that the variation of elastic moduli between separate regions (anterior, middle and posterior) of the skull were significantly different in cortical bone, but was not in trabecular bone. Elastic moduli measured in different orthotropic planes were also significantly different, with the moduli measured in the mediolateral direction consistently lower than that measured in either the anteroposterior or ventrodorsal direction. These findings demonstrate the significance of regional and directional variation in cortical bone elastic modulus, and therefore material properties in finite element models of the skull, particularly those of the rabbit, should consider the heterogeneous and orthotropic properties of skull bone when possible

Simultaneous Assessment of Homonymous and Heteronymous Monosynaptic Reflex Excitability in the Adult Rat

In order to successfully perform motor tasks such as locomotion, the central nervous system must coordinate contractions of antagonistic and synergistic muscles across multiple joints. This coordination is largely dependent upon the function of proprioceptive afferents (PAs), which make monosynaptic connections with homonymous motoneurons. Homonymous pathways have been well studied in both health and disease but their collateral fibers projecting to heteronymous, synergistic muscles receive relatively less attention. This is surprising given that PA collaterals have significant effects on the excitability of heteronymous motoneurons, and that their synaptic terminal density is activity dependent. It is likely that the relative lack of literature is due to the lack of a preparation which allows synergistic heteronymous pathways to be assessed in vivo. Here, we describe a method to simultaneously evoke homonymous and heteronymous (synergistic) monosynaptic reflexes (MSRs) and study their modulation by descending pathways in adult rats. Through stimulation of the medial plantar nerve, we were able to produce an H reflex in the intrinsic foot (IF) muscles of the hind paw with a latency of 10.52 ± 3.8 ms. Increasing the stimulus intensity evoked a robust signal with a monosynaptic latency (11.32 ± 0.35 ms), recorded in the ipsilateral gastrocnemius (Gs). Our subsequent analyses suggest that Gs motoneurons were activated via heteronymous afferent collaterals from the medial plantar nerve. These reflexes could be evoked bilaterally and were modulated by conditioning stimuli to the cortex (Cx) and reticular formation. Interestingly, cortical stimulation was equally efficient at modulating both ipsilateral and contralateral reflexes, indicating that cortical modulation of lumbar sensory afferents lacks the laterality demonstrated by studies of cortical muscle activation. This technique represents a novel, relatively simple way to assess heteronymous afferent pathways in normal motor control as well as in models of motor disorders where adaptive and maladaptive plasticity of PAs and descending systems affects functional outcomes

Regional variation in the mechanical properties and fibre‐type composition of the rat extensor digitorum longus muscle

Fibre‐type composition is heterogeneous, and distribution varies spatially in many muscles, indicating that there might be regional variation in recruitment and mechanical output. The rat extensor digitorum longus muscle is composed of predominantly fast‐twitch fibres and exhibits a gradient in phenotype, resulting in oxidative medial (areal composition 24.3% type I/IIa) and glycolytic lateral (92.4% type IIx/IIb) compartments. Here, we investigated the variation in mechanical performance between the medial and lateral compartments during isometric, isotonic and cyclical contractions. Isometric tetanic stress and force–velocity relationships were similar in both compartments, but isometric twitch kinetics were slower in the medial compared with the lateral compartment. The medial compartment also had a lower optimal cycle frequency for maximal net power generation (11 versus 15 Hz; P < 0.05) attributable to slower isometric kinetics, resulting in a lower level of activation and reduced net work generation at higher cycle frequencies, compared with the lateral compartment. The more oxidative, medial compartment had higher fatigue resistance, maintaining net power 26% longer than the lateral compartment. The predominant mechanisms underpinning the decrease in net power varied between the compartments, resulting from an increase in the work to extend the muscle and from a reduction in work during shortening in the medial and lateral compartments, respectively. Regional variation in mechanical performance and resistance to fatigue within a mixed muscle suggests that a differential recruitment pattern is likely during locomotion, with the medial compartment being used during slow‐speed locomotion and the lateral compartment during burst activities

The importance of capillary distribution in supporting muscle function, building on Krogh's seminal ideas

Krogh's Nobel prize for insightful studies into the physiology of capillaries heralded a revolution in understanding that continues today. The view of passive conduits has been replaced by capillaries recognised as a key element in haemodynamic control, offering both a site where changes in tissue demand are sensed and a driver of integrated vascular responses. In addition, the capillary bed is known to play an important role in metabolic, hormonal and immune homeostasis. Not surprisingly, therefore, microvascular dysfunction is a hallmark of many central and peripheral diseases, leading to widespread morbidity and mortality. Consequently, there is growing interest in how best to specifically target this organ-system by means of effective angiotherapies. Underpinning a lot of our current understanding of capillary physiology has been a recognition of functional heterogeneity among different microvascular beds. In addition, there is increasing awareness of the role that spatial heterogeneity plays in determining both physiological and pathological outcomes that has led to an appreciation that quality, rather than just quantity of microvascular supply is important. This has required a re-appraisal of the methods used to determine both the extent and topology of the capillary network, with the benefit of facilitating new ways of exploring dynamic regulation of capillary supply and its potential consequences

A novel simplistic fabrication technique for cranial epidural electrodes for chronic recording and stimulation in rats

Background: The demand for neuromodulatory and recording tools has resulted in a surge of publications describing techniques for fabricating devices and accessories in-house suitable for neurological recordings. However, many of these fabrication protocols use equipment which are not common to biological laboratories, thus limiting researchers to the use of commercial alternatives. New method: We have developed a simple yet robust implantable stimulating surface electrode which can be fabricated in all wet-bench laboratories. Results: Female Sprague-Dawley rats received epidural implantation of the electrodes over the fore and hind limb areas of their motor cortex. Stimulation of the motor cortex successfully evoked fore- and hind limb motor outputs. The device was also able to record surface potentials of the motor cortex following epidural stimulation of the spinal cord. Comparisons with existing methods: For stimulation of the motor cortex, often stiff stainless or copper wires are roughly tucked underneath the skull, with little accuracy of localization. While, commercially available devices utilize burr holes and screw electrodes. Our new electrode design provides us stereotaxic accuracy that was not previously available. Conclusion: We developed a chronic implantable electrode capable of being fabricated in all wet-labs, are robust, versatile and electrically sensitive enough for long-term chronic use. The simple and versatile electrode design provides scientific, economical and ethical benefits

Heterogeneity in form and function of the rat extensor digitorum longus motor unit

The motor unit comprises a variable number of muscle fibres that connect through myelinated nerve fibres to a motoneuron (MN), the central drivers of activity. At the simplest level of organisation there exist phenotypically distinct MNs that activate corresponding muscle fibre types, but within an individual motor pool there typically exists a mixed population of fast and slow firing MNs, innervating groups of Type II and Type I fibres, respectively. Characterising the heterogeneity across multiple levels of motor unit organisation is critical to understanding changes that occur in response to physiological and pathological perturbations. Through a comprehensive assessment of muscle histology and ex vivo function, mathematical modelling and neuronal tracing, we demonstrate regional heterogeneities at the level of the MN, muscle fibre type composition and oxygen delivery kinetics of the rat extensor digitorum longus (EDL) muscle. Specifically, the EDL contains two phenotypically distinct regions: a relatively oxidative medial and a more glycolytic lateral compartment. Smaller muscle fibres in the medial compartment, in combination with a greater local capillary density, preserve tissue O2 partial pressure (PO2) during modelled activity. Conversely, capillary supply to the lateral compartment is calculated to be insufficient to defend active muscle PO2 but is likely optimised to facilitate metabolite removal. Simulation of in vivo muscle length change and phasic activation suggest that both compartments are able to generate similar net power. However, retrograde tracing demonstrates (counter to previous observations) that a negative relationship between soma size and C-bouton density exists. Finally, we confirm a lack of specificity of SK3 expression to slow MNs. Together, these data provide a reference for heterogeneities across the rat EDL motor unit and re-emphasise the importance of sampling technique