Inducing neuron regeneration at the lesion site is thought to be the main barrier to functional recovery from spinal cord injury. Deleting the tumor suppressor gene PTEN (phosphatase and tensin homolog) resulted in substantial neuron regeneration and axonal sprouting, providing a method to overcome this barrier. PTEN deletion reactivates the mammalian target of rapamycin (mTOR) pathway, promoting enhanced axon regeneration. The functional locomotion recovery, however, still needs to be assessed in PTEN deleted mutants following spinal cord injury. Although neuron regeneration is a potential therapeutic approach in achieving functional recovery, there is no evidence indicating significant locomotion recovery in the presence of neuroregeneration following spinal cord injury.

In this study, we assessed locomotion recovery in Cre-mediated homozygous PTEN deleted mice following spinal cord crush injury. PTEN was conditionally deleted by tamoxifen induced Cre-mediated recombination system in transgenic mice resulted from crossing the SLICK-H mice with PTEN (loxP-loxP) mice. Following the laminectomy of T6-T7, each animal was subjected to a 20-second spinal cord crush.

Locomotion function was quantitatively assessed weeks 3 and 5 post-injury using Catwalk Gait Analysis to evaluate the effect of PTEN deletion in functional recovery.

The results indicated that PTEN deletion alone does not promote significant functional recovery. Thus, there are other intrinsic or extrinsic factors to neurons that contribute to locomotion recovery failure despite the induced axonal regeneration at the lesion site. For a potential therapeutic treatment, other strategies should also be considered in combination with PTEN deletion.

Introduction 

Promoting neuron regeneration at the lesion site appears to be the main obstacle in achieving functional recovery after SCI. Mature central nervous system (CNS) neurons were thought to lack the ability to generate new neurons until neurogenesis from mature neurons was demonstrated in discrete regions of the adult human brain.1 Following this observation, various approaches were used to induce neuron regeneration in spinal cord injury models to achieve functional recovery.2-5 The ability of neurons to regenerate following spinal cord injury is monitored by both the intrinsic capacity of neurons to regrow, and the presence of extrinsic inhibitory molecules in the extracellular environment.6-8 Neurons have a limited intrinsic ability to regenerate short-distance axonal sprouting. Therefore, enhancing the intrinsic regrowth ability of neurons is necessary for successful long-distance regeneration in spinal cord injury models.2,8 In previous studies, it was observed that deletion of the tumor suppressor gene PTEN (phosphatase and tensin homolog) in differentiated neurons results in neuronal hypertrophy and increased axonal growth.3-5 In a later study, it was demonstrated that PTEN deletion results in a substantial regrowth in the retinal ganglion cells at the lesion site soon after injury.9,10 PTEN deletion induces axon regeneration and sprouting by reactivating the mammalian target of rapamycin (mTOR) pathway which determines the regrowth ability of neurons.10,11 PTEN deletion was later examined in corticospinal neurons following spinal cord injury and the results showed a robust regeneration and sprouting of axons.12 However, functional locomotion recovery after spinal cord injury still needs to be assessed in PTEN deleted mutants. In this study, we assessed locomotion recovery in Cre-mediated homozygous PTEN deleted mice following spinal cord crush injury. PTEN was deleted using tamoxifen induced Cre-mediated recombination system to achieve neuron regeneration and axon sprouting after spinal cord injury.10,12 Cre-recombinase was fused with a mutated ligand-binding domain of the estrogen receptor (ER) which then was activated by synthetic ligand 4-hydroxy tamoxifen.13 The Cre-ERT expression is coupled with the expression of enhanced yellow fluorescent protein (EYFP) marker, and both genes are controlled by the tissue specific Thy1 promoter in SLICK-H transgenic mice.14 This promoter limits the expression of Cre to mature neuron tissues of CNS, therefore restricting the recombination to these tissues.14-15

Methods and Materials

All procedures were approved by the Animal Care Committee of the University of British Columbia, in accordance with the guidelines of the Canadian Council for Animal Care.

Transgenic Mice

A total of 25 female 8-9 month old mice were used in this study with n=15 homozygous conditional floxed PTEN (PTENloxP/loxP) and n=10 heterozygous PTENloxP/-. Both groups were heterozygous for CreERT2 expression coupled with the expression of an enhanced yellow fluorescent protein (EYFP); both genes regulated by two separate copies of Thy1 promoter.14 These transgenic strains were generated by primarily crossing C129S4-Ptentm1Hwu/J (homozygous PTEN(loxP-loxP)) strain with STOCK Tg(Thy1-cre/ERT2,-EYFP) HGfng/PyngJ strain (The Jackson Laboratory, Bar Harbor, ME). The resulting progeny was heterozygous for both PTENloxP-loxP and Thy1- creERT2/-EYFP genes. The progeny was then crossed with the original homozygous PTENloxP-loxP to generate the homozygous and heterozygous mice.

Tamoxifen Administration

Each mouse was injected with 80mg/kg of 4-hydroxy tamoxifen via intraperitoneal injection two weeks prior to spinal cord injury. They received one injection per day for 3 consecutive days.

Spinal Cord Crush injury

The procedure of T6/7 spinal cord crush is similar to what was previously described12,30 with modifications. All procedures were performed under aseptic conditions. The mice were anesthetised with the inhalant isoflurane. The skin of the back of each animal was shaved and disinfected, and they were given subcutaneous injection of 1 ml Lactated- Ringer’s with Buprenorphine (0.03mg/kg). Following the setup of the surgical plane, a laminectomy was performed to expose T6/T7 spinal cord segments. Fine tipped Dumont #5 forceps with custom tips ~200 μm in width were used. Altering the spacer, a precise closure distance was provided and checked under the microscope, and the spinal cord was laterally compressed using the forceps to a thickness of 0.3 mm and the compression was held for 20 seconds. Following the compression, the skin and muscle tissues were closed with sutures, and the animals were allowed to recover in an incubator (35ͦC) before returning to their home cage. The mice received Ringer’s solution for hydration (1mL) and Buprenorphine (0.03mg/Kg) twice a day for the next 2 days to alleviate pain and sunflower seeds were added to their diet for a week. Animal weights were monitored daily for a week post-surgery and their bladders were expressed daily until the mice reached normal micturition. One PTENloxP/loxP animal was found dead in its cage several days after the surgery.

Catwalk Gait Analysis

Catwalk Gait Analysis was performed to assess the functional recovery of the groups and quantify their hindlimb locomotion.16 The test was carried out once prior to the injury to establish a baseline value. Catwalk Gait Analysis was delayed until week 3 following injury, allowing the animals to regain plantar placement with weight support, and the test was conducted at 3 and 5 weeks post-injury. To assess gait patterns, each animal ran across a transparent platform (5.2 cm wide and 60 cm long) with opaque and black walls in a darkened room and were filmed from underneath while crossing the platform with a high-definition camera (Sony, HDR-HC1) illuminated with the light source below the platform. The light is internally reflected in the glass floor except for points where a paw touches the glass. It then exits the floor and illuminates the contact area, generating a footprint.17 Mice were trained to run from a foreign cage, across the walkway and back to their home cage prior to testing. At each time point, four runs for each animal were recorded from underneath the glass. The three most consistent runs were chosen within the four runs and analysis was performed on 3 normal step sequence patterns in each uninterrupted run. If all runs were consistent then the last run was automatically excluded. The hindlimb stride length (the distance between successive placement of the same paw), hindlimb print area (the total area of the complete paw print) were measured, as was base of support which defines the average width between the centers of the hind paws, and step sequence duration indicating the speed in crossing the platform.16

Statistical Analysis

Sigma Stat 3.0.1 was used for the statistical tests. All data were presented as mean ± standard error of the mean (SEM). As the data were not normally distributed, the nonparametric Mann-Whitney U Tests was used for the stride length, print area and the step sequence duration parameters. Two-way repeated measures ANOVA was used to analyze the measures obtained for the base of support, with the significance level set at p < 0.05 for all tests.

Results

One animal from the control group failed to cross sufficiently at the time points post-injury, thereby was excluded from analysis. At the time of statistical analysis, the gait parameters of 14 animals in the PTENloxP/loxP group and 9 animals in the PTENloxP/- group were statistically analysed.

Base of Support

Hindlimb base of support increased at week 3 post-injury followed by a decrease at week 5 in both groups(Fig 1A). However, there was no significant difference in base of support between the groups over time (p(3w)=0.550; p(5w)=0.691; p> 0.05, data not shown).

Step Sequence Duration

The average step sequence duration was measured to estimate the speed of the individual groups. The step sequence duration for mice in the control group exhibited a decrease at week 3 followed by an increase at week 5 (Fig 1B). However, the PTENloxP/loxP mice appeared to cross the platform faster over time post-injury. Overall, no significant difference was observed in PTEN deleted mice compared to the control group (p(3w)=0.850; p(5w)=0.950; p> 0.05, data not shown).

DISCUSSION

This study characterises the locomotion recovery in PTEN deleted adult mice following spinal cord injury. The functional recovery of the hindlimbs was assessed using Catwalk Gait Analysis by measuring changes in locomotion parameters such as stride length, print area, base of support and the step sequence duration at time points week 3 and week 5 post-injury. Despite the promising evidence showing that PTEN deletion can enhance neuron regeneration following SCI, our results in the Catwalk Analysis of different gait parameters indicate that PTEN deletion did not lead to a significant locomotion recovery following spinal cord injury. All assessed gait parameters in this study did not exhibit a significant difference between the PTENloxP/loxP and PTENloxP/-groups over time. Although the Catwalk Gait Analysis provides a quantitative and relatively sensitive method for objective assessment of recovery, the variable gait speed at which the animal chooses to locomote is a specific limitation of this method. The efficiency of neuron regeneration and sprouting is also to be determined in the future using EYFP (enhanced yellow fluorescence protein) labeling and immunostaining to ascertain regeneration at the lesion site. However, it has been previously demonstrated that PTEN deletion leads to substantial regrowth of neurons at the lesion site.9,12 It has also been demonstrated that Cre-mediated PTEN deletion occurs with high efficiency in many regions of CNS of adult SLICK-H transgenic mice in addition to spinal cord.14 PTEN deletion causes significant behavioral abnormalities in mice such as decreased learning, decreased social interaction, abnormal anxiety, seizures and autistic-like behaviors.4 Both homozygous and heterozygous mice appeared hyperactive and exhibited inappropriate responses such as exaggerated reaction to sensory stimuli and anxiety-like behaviors following PTEN deletion. These side effects of PTEN deletion may hinder the assessment and comparison of functional locomotion in this study resulting in the negative outcomes. Technologies that restrict PTEN deletion mainly in the neurons affected by the lesion can eliminate the consequences of PTEN deletion in the other regions of the CNS resulting in more accurate outcomes in locomotion recovery.

The extracellular environment is a critical determinant of neuron regeneration and axonal sprouting. Extrinsic inhibitory molecules in the extracellular environment limit neuron regeneration and sprouting through the lesion site following the deletion of PTEN.7,8 These inhibitory molecules also decrease the plasticity of newly generated axons.18 Following the injury, formation of glial scar as a result of recruitment of microglia, oligodendrocyte precursors, meningeal cells, and astrocytes at the lesion site can inhibit axonal regeneration.8,19,20 The formation of glial scar is beneficial in terms of isolating the lesion site and minimising the overwhelming inflammatory response which eventually limits tissue damage.20 However, the activated immune cells at the lesion site lead to axonal regenerative failure by generating a physical barrier at the lesion site and expression of extrinsic

inhibitory factors.19 Therefore, it is important to consider modulating both extrinsic and intrinsic factors to enhance neuron growth and plasticity to achieve functional recovery following SCI. To achieve a functional locomotion recovery, the newly generated axons need to establish a functional integration into the pre-existing circuits and maintain synaptic connection with their specific targets.

Spontaneous functional recovery can be promoted in incomplete spinal cord injury models by the following: synaptic plasticity in intact neurons, and by anatomical plasticity as a result of new axonal sprouting from the uninjured neurons surrounding the lesion site.21,22 Newborn neurons from adult neurogenesis in discrete regions of CNS also exhibit enhanced synaptic and anatomical plasticity, and the magnitude can be modified in response to experience.23-26 Therefore, treatments that enhance neuron plasticity have an important contribution to functional recovery.27 Studies revealed that the newly generated axons from PTEN deleted neurons also have the capacity to reform synapses in regions distal to the lesion site at corticospinal tract.12 Rehabilitation treatments and exercise are widely used methods to increase locomotor function by enhancing synaptic plasticity and increasing the excitability of CNS neuronal networks.21,22,28,29

Conclusion

Despite the promising evidence showing that PTEN deletion can enhance neuron regeneration following SCI, this study revealed no correlation between PTEN deletion and functional locomotion recovery. The results from Catwalk Gait Analysis indicate that PTEN deletion alone does not promote significant functional locomotion recovery over time. Other intrinsic and extrinsic factors alter the effect of PTEN deletion on functional locomotion recovery. Thereby, PTEN deletion must be used in combination with other strategies that account for other barriers to neuron regeneration to result in better recovery outcomes.

Acknowledgments

I am very grateful to Dr. Wolfram Tetzlaff for giving me the opportunity to perform this study at Blusson Spinal Cord Centre. I would like to give special thanks to my mentor and supervisor, Dr. Brett Hilton, who guided me throughout the study. I would also like to thank Dr. Jie Liu for performing the surgeries, Dr. Greg Duncan for aiding me during the Catwalk Gait Analysis and Dr. Peggy Assinck for helping me during the statistical analysis.

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