Acute Intermittent Hypoxia and Body Weight Supported Treadmill Training for Incomplete Spinal Cord Injury Patients

Spinal cord injury (SCI) interrupts descending synaptic pathways from brainstem premotor neurons to spinal motor neurons, thereby paralyzing muscles below the neurological level. In recent years, considerable evidence has demonstrated that acute intermittent hypoxia (AIH) elicits plasticity in the spinal cord and strengthens spare synaptic pathways which is expressed as respiratory and somatic functional recovery in animals and humans suffering from incomplete SCI. The fundamental hypothesis guiding this project is that AIH-induced motor plasticity can be "harnessed" to improve walking capacity in incomplete SCI patients, classified as C and D categories according to International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI). The inclusion criteria include patients \> 18 years-old, with traumatic or non-traumatic, non-progressive incomplete SCI, onset \> 6 months, neurological level C5-T12, with walking ability with or without assistive devices, without joint contractures, orthopedic injuries, osteoporosis, cutaneous lesions, cardiopulmonary complications and a body weight below 150 Kg. A randomized, triple-blind, placebo-controlled parallel design study will be done including 100% of patients fulfilling the criteria. Participants will receive repetitive acute intermittent hypoxia (rAIH: 15 episodes of 90 second 9% inspired oxygen interspersed with 90-second normoxia) or repetitive continued normoxia (rSham: 21% inspired oxygen) combined with 45 minutes body weight-supported treadmill training on 5 consecutive days and then three times per week for 3 weeks. Primary outcome measurement will be the 10-meter walking test. Secondary outcome measurements include the 6-minute walking test, timed up and go test, body/weight load, modified ashworth scale and visual analog scale. All outcomes will be measured before beginning the protocol (baseline), after five days of AIH/Sham (D5), weekly up to the end of the study (W2-W4), and a post-study follow-up for 2 weeks (F1-F2). Aditionally, cognitive assesment before and after the study will be performed using the "Figura compleja de Rey-Osterrieth" and the "Test de aprendizaje verbal España Complutense (TAVEC)". Repetitive AIH and body weight-supported treadmill training may represent a novel, safe, and noninvasive potential therapy to partially restore walking function in incomplete sub-acute and chronic SCI patients, a population with limited, if any, potential for improved function.

Currently, there is no "cure" for spinal cord injuries (SCI). Although the scientific understanding of central nervous system (CNS) regeneration has advanced greatly in the past twenty years, there are still many unknowns with regard to inducing successful regeneration, especially with chronic SCI. A more realistic approach, based on currently available knowledge, to improve the quality of life for a large proportion of the paralyzed population may be to develop treatments that elicit partial functional recovery based on neuroplastic potential of spared neural pathways. In this scenario, acute intermittent hypoxia (AIH) enhances the inherit capacity for neuroplasticity and strengthens surviving synaptic inputs onto spinal motorneurons, which trigger functional recovery following SCI in rats and humans AIH-induced neuroplasticity has been extensively studied in phrenic motor nuclei (C3-C5) through phrenic nerve recording preparations (Mitchell, 2007). Briefly, moderate AIH (3, 5-min hypoxic episodes; PaO2 35-45 mmHg; 5-min intervals) elicits phrenic long term facilitation (pLTF), a type of memory present in the cervical spinal cord. The mechanism of AIH-induced pLTF is that episodic hypoxia activates raphe serotonergic neurons that project to phrenic motor nuclei. Spinal serotonin release during hypoxic episodes subsequently activates serotonin type 2 (5-HT2) receptors coupled to Gq protein on or near phrenic motor neurons, and initiates intracellular cascades that underlie pLTF . pLTF requires spinal 5-HT2 receptor activation , new synthesis of brain-derived neurotrophic factor (BDNF) and activation of its high-affinity receptor tyrosine kinase (TrkB) followed by ERK MAP kinase signaling. Although downstream signaling events from ERK are less clear, it is speculated that glutamate receptors are phosphorylated, increasing glutamatergic transmission and perhaps insertion within phrenic motor neurons, thereby establishing LTF.

Longer time domains of AIH, for example, daily acute intermittent hypoxia (i.e. dAIH, 10 episodes per day, 7 days) have shown to strengthen synaptic pathways to spinal motorneurons and increase respiratory and locomotor recovery after cervical SCI in unanesthetized rats. This functional improvement is accompanied by increased BDNF and TrkB levels within cervical (C7) motor nuclei innervating the forelimb. Although the detailed mechanisms of the functional recovery in somatic thoracic or lumbar motorneurons have not been verified, it has been proposed that the same serotonin-dependent mechanisms facilitate motor output in respiratory and non-respiratory motor nuclei.

The use of dAIH to improve limb function in humans with incomplete, chronic SCI has shown promising results. A single presentation of AIH (15, 1-minute episodes of 9% O2 alternating with 1-minute of 21% O2) in incomplete, chronic (\>1 year) spinal cord injury patients, classified as C or D according to the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI), increases the ability to voluntarily generate plantar flexion 4 h post-hypoxia. In a randomized, double-blind, placebo-controlled, crossover design study, the impact of daily AIH (15 episodes per day, 90 sec 9% O2, 60 sec normoxic interval, 5 consecutive days) combined with walking training was studied in 19 chronic, incomplete SCI patients (ISNCSCI D). Daily AIH alone increased walking speed 18% three days after treatment (10 m walk test); whereas dAIH combined with walking training improved both walking speed and distance (37%) after 5 days and 1 week post-dAIH. Importantly, no changes in cognitive function was observed after dAIH, suggesting that this moderate dose of AIH is safe in humans.

Although dAIH (5 consecutive days of AIH) has demonstrated beneficial effects in incomplete SCI patients, its effect last only up to one week; therefore, is important to design extended protocols maintaining the initial functional effect of dAIH over time. Repetitive AIH (rAIH) consisting of AIH three times per week (3×wAIH) for 10 weeks have demonstrated to increase respiratory function and maintain the increased functional effect elicited by dAIH in unanesthetized rats. Moreover, rAIH increases the expression of key molecules involved in AIH-induced spinal plasticity in unanesthetized rats. Therefore, repetitive AIH may represent a safe and effective strategy to enhance functional recovery after chronic incomplete spinal cord injuries.

The protocol of intermittent hypoxia proposed in this project corresponds to a moderate dose of intermittent hypoxia, which is the equivalent of climbing a mountain at 5000 meters altitude. Abundant literature has demonstrated that moderate AIH (≥ 9% O2, \< 15 cycles/day) have several multi-systemic beneficial effects: reduces arterial hypertension, strengthens innate immune responses, reduces inflammation, reduces body weight, increases aerobic capacity, improves glucose tolerance, increases bone mineral density, enhances spatial learning and memory, rescues ischemia-induced memory impairment, reduces symptoms of depression, improves post-ischemic recovery of myocardial contractile function, and increases respiratory capacity in chronic obstructive pulmonary disease. Moreover, moderate repetitive AIH improves respiratory and somatic function after SCI, without adverse consequences such as hypertension, neuronal cell loss and/or reactive gliosis or systemic inflammation. Therefore, the potential beneficial effects of AIH are not only limited to spinal cord injuries but include a wide scope of clinical conditions.

Combinatorial therapies, one of them being an activity-based training, can augment plasticity after incomplete SCI. In rats with incomplete SCI, dAIH combined with ladder walking leads to near complete recovery of ladder walking ability. Moreover, dAIH and overground walking improve walking speed and distance in incomplete SCI patients ISNCSCI D. Research studies in animals and humans have found that retraining after SCI using the intrinsic physiologic properties of the nervous system can facilitate the recovery of function. This potential for retraining is based on activity-dependent plasticity driven by repetitive task-specific sensory input to spinal networks.

The most prominent and well-developed activity-based therapy (physical rehabilitation) to date is locomotor training. The fundamental principles of locomotor training are built on the premise of robustly approximating the sensorimotor experience of walking through repetitive practice including: 1) maximize load bearing by the lower extremities and minimize load bearing by the upper extremities, 2) optimize the sensory cues for walking, 3) optimize the kinematics (i.e., trunk and extremities) for each motor task, and 4) maximize recovery strategies and minimize compensatory mechanisms. The fundamental mechanisms supporting this intervention have been derived largely from studies conducted in spinalised animals. Specifically, treadmill training increases axonal regrowth and collateral sprouting proximal to the lesion site in mice (Goldshmit et al., 2008), phosphorylation of Erk1/2 in the motor cortex as well as the spinal cord injury area (Oh et al., 2009), expression of brain-derived neurotrophic factor (BDNF) in the spinal cord, ameliorates muscle atrophy in moderate contused SCI rats, and alters properties of spinal motor neurons. Body weight-supported treadmill training (BWSTT) is based on optimizing sensory inputs relevant to step training, repeated practice, and possible optimization of neuroplasticity. Uncontrolled studies in acute and chronic SCI patients show within-subject improvements in walking ability using BWSTT. Investigators propose that BWSTT therapy provide a behavioral therapy that independently supports positive outcomes.

AIH combined with body weight-assisted training represents a simple and safe, non-pharmacological method for enhancing neuroplasticity in the spinal cord and thus, improving walking function in patients with incomplete spinal cord injuries. At the cellular level, both BWSTT and AIH increase the expression of BDNF. BDNF has a wide repertoire of neurotrophic and neuroprotective properties in the CNS and the periphery; namely, neuronal protection and survival, neurite expression, axonal and dendritic growth and remodeling, neuronal differentiation and synaptic plasticity such as synaptogenesis in arborizing axon terminals, and synaptic transmission efficacy. Thus, BWSTT may serve as a catalyst in tandem with repetitive AIH that when combined develop an even better response.

Currently, there are no approved therapies for chronic SCI; therefore, the approach represents a promising new strategy to enhance function in patients with sub-acute and chronic SCI, where the potential for further functional gains is limited.

Investigators propose a triple blind (patients, outcome assessors and stadistician) randomized, placebo-controlled study testing the combined effect of intermittent hipoxia and body weight-supported treadmill training in incoplete spinal cord injury patients.

Eligibility Criteria:
Inclusion Criteria:

1. Patients ≥ 18 years-old from "Instituto Teletón Santiago" and "Hospital Clínico Mutual de seguridad".
2. C5 to T12 spinal cord injury, classified as ISNCSCI grades C and D
3. Traumatic and non-traumatic, non-progressive lesions
4. Onset \> 6 months
5. Ability to ambulate with or without assistive devices
6. Ability to follow verbal or visual commands
7. Signed informed consent

Exclusion Criteria:

1. Orthopedic injuries that are unstable
2. Osteoporosis with high risk of pathological fracture
3. Cutaneous lesions and/or pressure ulcers
4. Joint contractures
5. Cardiopulmonary diseases
6. Body weight exceeding 150 Kg