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Logo of neuMary Ann Liebert, Inc.Mary Ann Liebert, Inc.JournalsSearchAlerts
Journal of Neurotrauma
 
J Neurotrauma. 2011 March; 28(3): 479–492.
PMCID: PMC3119327

Intermittent Fasting Improves Functional Recovery after Rat Thoracic Contusion Spinal Cord Injury

Abstract

Spinal cord injury (SCI) often results in a loss of motor and sensory function. Currently there are no validated effective clinical treatments. Previously we found in rats that dietary restriction, in the form of every-other-day fasting (EODF), started prior to (pre-EODF), or after (post-EODF) an incomplete cervical SCI was neuroprotective, increased plasticity, and promoted motor recovery. Here we examined if EODF initiated prior to, or after, a T10 thoracic contusion injury would similarly lead to enhanced functional recovery compared to ad libitum feeding. Additionally, we tested if a group fed every day (pair-fed), but with the same degree of restriction as the EODF animals (~25% calorie restricted), would also promote functional recovery, to examine if EODF's effect is due to overall calorie restriction, or is specific to alternating sequences of 24-h fasts and ad libitum eating periods. Behaviorally, both pre- and post-EODF groups exhibited better functional recovery in the regularity indexed BBB ambulatory assessment, along with several parameters of their walking pattern measured with the CatWalk device, compared to both the ad-libitium-fed group as well as the pair-fed group. Several histological parameters (intensity and symmetry of serotonin immunostaining caudal to the injury and gray matter sparing) correlated with functional outcome; however, no group differences were observed. Thus besides the beneficial effects of EODF after a partial cervical SCI, we now report that alternating periods of fasting (but not pair-fed) also promotes improved hindlimb locomotion after thoracic spinal cord contusion, demonstrating its robust effect in two different injury models.

Key words: calorie restriction, every-other-day fasting, functional recovery, intermittent fasting, spinal cord injury

Introduction

The majority of human spinal cord injuries are anatomically incomplete, yet aside from physical rehabilitation, there are no clinically validated treatments available to target the injured spinal cord and improve functional recovery. Pre-clinical research focuses on the prevention of secondary degeneration, and the enhancement of endogenous neural plasticity in the spared spinal cord, as well as axonal regeneration (Bareyre et al., 2004; Park et al., 2004; Ramer et al., 2005).

Ideally, therapeutic interventions in acute SCI should provide neuroprotection as well as increased plasticity and axonal regeneration (Ramer et al., 2005). Dietary restriction (DR), with adequate nutrition, which is well established to increase lifespan in a diverse set of species, could potentially represent such a versatile broad-spectrum approach. There are two main forms of DR: calorie restriction (CR), in which daily calorie intake is reduced 20–40%, and intermittent fasting, which is usually carried out by fasting the animals for 24 h, followed by 24 h of ad libitum access to food. Prophylactic intermittent fasting (e.g., every-other-day fasting [EODF]) can protect neurons against injury by enhancing endogenous cellular stress responses and energy metabolism, by dampening oxygen free radical formation and inflammation, and activating cell survival pathways, as well as by increasing growth factor expression and axonal plasticity (Fontan-Lozano et al., 2007; Mattson et al., 2002, 2005; Plunet et al., 2008).

DR has biochemical and functional benefits in a wide range of animal injury/disease models, including seizures (Bough et al., 1999, 2003), excitotoxicity (Anson et al., 2003; Bruce-Keller et al., 1999; Sharma and Kaur, 2005), stroke (Yu and Mattson, 1999), Parkinson's disease (Maswood et al., 2004), Huntington's disease (Bruce-Keller et al., 1999; Duan et al., 2003), and Alzheimer's disease (Halagappa et al., 2007; Mouton et al., 2009; Patel et al., 2005; Qin et al., 2006; Wang et al., 2005; Wu et al., 2008; Zhu et al., 1999).

In most studies the benefits of DR were seen in chronic disease models, or in cases of acute insults, with DR pretreatment of several weeks to multiple months prior to insult. More recently and in contrast to this previous work, a 24-h fast initiated after a moderate traumatic brain injury (TBI) also resulted in improved functional outcome in the Morris water maze, and increased neuroprotection (Davis et al., 2008). Similarly, we reported the effectiveness of EODF, the most widely used form of intermittent fasting, when started after a cervical SCI (Plunet et al., 2008). After a unilateral crush of the dorsolateral funiculus of the cervical spinal cord, the EODF-treated rats performed significantly better than the ad-libitum-fed group in a forelimb usage (cylinder) test, as well as in an irregular ladder crossing test. The loss of neurons and gray matter tissue in the vicinity of the lesion was significantly reduced by treatment with EODF, and this correlated with behavioral outcomes. Additionally, we observed an increase of sprouting in the intact corticospinal cord tract, which also correlated with functional outcome. These observations suggest that intermittent fasting might be a potential treatment strategy after acute SCI, that could improve the quality of life in individuals with SCI.

In light of a recently published survey, which revealed that contusion injuries are deemed to be the most clinically relevant model (Kwon et al., 2010), we decided to test the efficacy of EODF after a thoracic spinal cord contusion injury. Data from this widely used model would allow us to compare the efficacy of EODF to other pre-clinical treatments. Furthermore, we included a pair-fed control group to determine whether daily calorie restriction diets exert similar beneficial effects as alternate days of fasting (EODF). This addresses the question of whether the positive results of EODF could be due to the general 20–30% reduction in calories typically observed in rats on EODF (Martin et al., 2007; Wan et al., 2003), or is something more specific to the 24-h fasting periods followed by 24 h of overfeeding. Some patients with SCI may prefer daily caloric restriction over alternate-day fasting regimens.

Methods

All procedures involving animals were approved by the Animal Care Committee of the University of British Columbia (UBC), in accordance with the guidelines of the Canadian Council for Animal Care and the National Institutes of Health. All efforts possible were made to reduce the number of animals used and to minimize pain.

Animals and general housing conditions

Sprague-Dawley rats (UBC breeding facility/Charles River Breeding Laboratories) were housed in the animal facility at the UBC with room temperature controlled at 21–22°C, and an artificial 12-h:12-h light:dark cycle (lights off at 8:00 pm). Upon arrival, the animals were group housed (n=3–4 per cage), and given food (LabDiet-5001; Purina Mills, Brentwood, MO) and water ad libitum until the start of the experiment. Each cage was supplied with housing tubes.

Feeding regimen at the start of the experiment

Before injury the animals were randomized into four different groups: (1) ad libitum (AL) group (n=12), which continued to have access to food ad libitum (but did not initiate eating until 4–8 h after surgery); (2) post-EODF group (n=12), starting a 24-h fast immediately after injury, then alternating between 24 h of feeding with free access to food and water, and 24 h of fasting with only access to water, and this alternating sequence of feeding/fasting continued throughout the rest of the experiment; (3) pre-EODF group (n=13), which started EODF 3 weeks prior to injury and continued this feeding regimen for the remainder of the experiment; or (4) pair-fed group (n=12), which were fed daily, but were matched for total food consumption with the EODF groups, starting 3 weeks prior to injury and continuing throughout the rest of the experiment (see Fig. 1 for a timeline of the experiment).

FIG. 1.
Timeline of the experiment. Both pair-fed and pre-EODF animals started their restricted diet 3 weeks prior to the scheduled spinal cord injury (SCI). The post-EODF group started its diet after the SCI. Baseline testing was performed prior to the injury. ...

Feeding and body weight monitoring

Daily food intake and the animals' weights were measured for all groups for the first 14 days after injury, and every 8 days after that. Pre-weighed food was provided in the food hopper of their home cage at 8:00 am (unless a fasting day for the EODF groups), and leftover food was weighed 24 h later. Daily food intake was estimated by cumulative food intake in a given cage divided by the number of animals in that cage. UBC animal care regulations do not allow single housing of rats simply for the estimation of food intake.

Thoracic spinal cord contusion injury

The rats were anesthetized with a ketamine/rumpon mixture (70 mg/10 mg per kg IP, respectively). After inducing a surgical plane of anesthesia, the incision site was shaved and disinfected with alternating povidone-iodine scrub and 70% alcohol. To prevent dehydration, 10 mL lactated Ringer's was injected (SC), and an injection of buprenorphine (0.03 mg/kg SC) was given to reduce pain. Lubricant eye ointment was applied to the eyes to avoid desiccation. Lidocaine containing epinephrine (0.1 mL) was injected at the incision site (IM) as a local anesthetic and to reduce bleeding.

The T10 lamina was removed with a pair of fine rongeurs to visualize the spinal cord. Following the laminectomy, the spine was stabilized by clamping the dorsal processes one segment above and below the lesion site. The rats received a spinal contusion injury utilizing the Ohio State impactor device (The Ohio State University, Columbus, OH), with a set displacement of 1.5 mm. Animals that received a contusion force above 250 kilodynes were excluded from the experiment. Following injury the rats were placed in a temperature-controlled incubator (37°C) until the animals were fully awake and moving. To prevent dehydration and post-operative pain, animals received 10 mL of Ringer's lactate solution and buprenorphine (0.05 mg/kg, 2 times/day, for a total of 2 days post-injury). The surgeon (J.L.) was blinded to the randomization of the rats.

Behavioral testing

Baseline (pre-operative) behavior was tested using three different behavioral tests: (1) the irregularly-spaced horizontal ladder, (2) the CatWalk, and (3) the Basso-Beattie-Bresnahan (BBB) open field assessment. All behavioral testing took place on a feeding day for the EODF animals to control for potential differences in motivation, and no food rewards were used in any of the behavioral testing (Plunet et al., 2008, 2010). Behavioral testing of the animals did not start until at least 4 h after the animals had access to food for the day. It was noted that after a 24-h fast the EODF animals gorged themselves during the first couple of hours, and had an extended abdomen, and gained the majority of their feeding day weight gains in those first few hours (approximately 75% of their typical feeding day weight gain of 30–40 g). Additionally, by performing behavioral testing on feeding days, it allowed the behavioral investigators to remain blinded to the treatment groups (the video analysis of the behavioral data on the ladder and the CatWalk was also done blinded to treatment group).

Irregularly-spaced horizontal ladder

The rats were trained to walk across a 110-cm-long horizontal ladder motivated by their home cage at the end. The ladder was 15 cm wide and consisted of 2-mm-diameter metal rungs. The scoring was limited to the middle 60-cm portion of the ladder, where the spacing between the rungs was varied, ranging from 1.75 to 4.0 cm. Each week the pattern of rungs was changed so the animal could not learn the sequence of rungs (Metz and Whishaw, 2002). Each animal crossed 7–8 times, and the runs were recorded by a high-definition camcorder. The hindlimb error rate was generated from frame-by-frame video replay, and was expressed as the percent error of the steps taken. A hindlimb foot-slip error/miss was designated when the animals completely missed the rungs and the hindlimb slipped noticeably below the rung's level; the error rate was expressed as a percentage of errors. The horizontal ladder was tested pre-injury, and at 50, 66, and 72 days post-injury (i.e., well after the rats had regained weight-supported stepping).

CatWalk gait analysis

Footprints of animals crossing an acrylic glass runway were marked using frame-by frame video replay of 4–6 uninterrupted crossings per animal (CatWalk; Noldus Information Technology, Wageningen, The Netherlands). Animals were tested on the CatWalk before surgery, and at 22, 36, 50, 64, and 70 days after injury.

The regularity index (RI) is a measurement of interlimb coordination. It is calculated from the number of normal step sequences (based on all patterns of all four limb placements), and the total number of steps. An RI of 100% represents perfect coordination (Hamers et al., 2001). In pre-operative testing 20.4% of our animals did not average a perfect 100% regularity index, with the lowest one averaging 96.01%. Our pre-operative RI percent averages were consistent with previous research using the CatWalk device (Hamers et al., 2001; Van Meeteren et al., 2003).

Typically, during coordinated walking as seen in uninjured animals the swing duration of forelimbs and hindlimbs are similar. Hence, subtracting the hindlimb swing duration from the forelimb swing duration yields a number close to zero (see Fig. 5A), and indeed this value was never smaller than −0.00338 sec, and usually was slightly positive. After injury, swing duration timing becomes disrupted, with longer duration of the hindlimb swing phase compared to the forelimb, typically resulting in a negative value. Similarly, in uninjured animals both the forelimb and hindlimb stride lengths were nearly identical, and hence subtracting the hindlimb stride length from the forelimb stride length yields a value near zero, but averages ~2.0 mm (see Fig. 5B). The largest difference we observed for any individual animal prior to injury was −3.42328 mm. However, after thoracic injury rats take smaller forelimb strides compared to their hindlimb stride, which results in large negative differences for most animals.

FIG. 5.
EODF improved swing duration differences between forelimbs and hindlimbs, as well as stride length differences. (A) After injury, swing duration timing is altered, resulting in a longer duration of the swing phase in the HL compared to the FL (FL – ...

Modified BBB open field assessment

Hindlimb locomotion was scored by the open-field locomotor scale described by Basso, Beattie, and Bresnahan (BBB; Basso et al., 1995, 1996) with some modifications. Briefly, the BBB is a 21-point scale (0=complete hindlimb paralysis and 21=normal locomotion), in which rats are scored based on hindlimb movements made in an open field during a 4-min interval while freely exploring the surroundings. The animals were tested prior to injury, and at 2, 8, 14, 22, 36, 50, 64, and 70 days post-injury.

The RI, obtained from the CatWalk device (Hamers et al., 2001, 2006), an objective measurement of coordination, was used to determine the coordination criteria of the open-field score from 22 days post-injury onward (after the animals were performing weight-bearing walking and were tested on the CatWalk device; Deumens et al., 2006; Koopmans et al., 2005, 2009). For an animal to be designated as occasional coordination, the animal had to perform at least one CatWalk run at 100% on the RI; for frequent coordination the animal had to average 95% RI or more, and have more than one run of 100%; to be considered as consistent coordination, individual animals had to have an average RI of 95% or above, and with at least half of the runs being at 100% RI. (Note: The RI percentage is not the same as the percentage of coordinated runs as traditionally assessed in the BBB.)

Histology

Animals were anesthetized with an overdose of sodium pentobarbital (107 mg/kg IP; Bimeda-MTC Animal Health Inc., Cambridge, Ontario, Canada), and perfused transcardially with 0.12 M PBS (0.43% NaCl, pH 7.4), followed by a solution of 4% paraformaldehyde (in 0.12 M PBS buffer, pH 7.4). The spinal cord was removed and cryoprotected overnight in 24% sucrose solution (in 0.12 M PBS). A 1.5-cm-long segment of the thoracic spinal cord containing the injury site was frozen for serial cross sectioning, as well as the lumbar enlargement, which was cross-sectioned for subsequent serotonin transporter (SERT) immunostaining caudal to the injury site. These regions of the spinal cord were cut on a cryostat at 20 μm thickness, serially collected, and frozen at −80°C until further use.

Lesion size and sparing analysis

The amount of spared white matter was measured on cross-sections 400 μm apart stained with eriochrome cyanine. A Zeiss Axioskop microscope with a 5×objective was used to capture images of the stained cross-sections ranging from 2800 μm rostal to 2800 μm caudal to the epicenter (as determined by the section with the least amount of spared white matter) of the injury for each animal. The rim of spared white matter was manually traced along with the amount of spared gray matter and quantified with SigmaScan Pro version 5.0 (Systat Software, Inc., Chicago, IL).

Immunofluoresence

For immunohistochemistry, sections were rehydrated in 0.01 M PBS (0.72% NaCl, pH 7.4) for 10 min and blocked with 10% NDS (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in 0.01 M PBS containing 0.1% Triton X-100 (PBS-T) for 30 min. Thereafter sections were incubated with primary antibodies against rabbit anti-SERT (1:500; ImmunoStar, Inc., Hudson, WI) diluted in PBS-T overnight at room temperature. After washing the slides three times for 5 min each (PBS), the sections were incubated with Cy3-conjugated donkey anti-rabbit IgGs (1:500; Jackson Immunoresearch Laboratories) in PBS-T for 2 h. After an additional washing step, the slides were cover-slipped with Fluoromount G (Southern Biotechnology, Birmingham, AL).

Quantification of raphe-spinal serotonergic fibers

From each animal, serial cross-sections spaced 400 μm apart starting at the rostral end of the lumbar enlargement (distal to the injury) were stained with SERT antibody. Digitized images of anti-SERT–stained sections (200×magnification) were captured and analyzed using Sigma Scan Pro 5. The total intensity per area (μm2) was analyzed, providing a density measurement (a.u). We analyzed the gray matter of the ventral horns (Rexed laminae VII, VIII, and IX), along with a more discrete measurement of Rexed lamina IX of the ventral gray matter. We analyzed each side of the ventral gray matter lamina IX separately to obtain a symmetry score by dividing the lowest scoring side by the highest (perfect symmetry would result in a symmetry score of 1.0).

Statistical analysis

For behavioral outcomes a two-way repeated-measures analysis of variance (ANOVA) (time and behavior) was used to compare the four groups, followed by a one-way ANOVA at each time point, and if significance was found, it was followed up by a Bonferroni post-hoc test (p<0.05, two-tailed). For histological data a one-way ANOVA was used and followed-up with a Bonferroni post-hoc if warranted. Pearson's correlations were calculated to examine relationships between functional behavioral outcomes (the average of each behavior over the last three testing times, on days 50, 64, and 70), along with the histological outcomes and behavioral results, also using the last three testing time points (r2 and p values are reported). All data are expressed as average±standard error of the mean (SEM). All tests of significance performed were two-tailed.

Results

An overview of the timeline of the experiment is presented in Figure 1.

Animal weight and food consumption

As expected the calorie-restricted groups consumed a lower total amount of food than the ad libitum eating (AL) control group, indicating that the restriction on the fasting day was not fully compensated on the eating days when food was offered ad libitum: post-EODF group (78.04±0.77%), and pre-EODF group (70.73±2.46%; p<0.05 by one-way ANOVA; Fig. 2A). This equates to approximately 22% and 29% calorie restriction for the two EODF groups. In an effort to match the food intake in the pair-fed group, we compromised and restricted it by 25%, at a level in between the two EODF groups.

FIG. 2.
Animal food intake and weight. Animals in the restricted diet groups consumed fewer calories than the ad libitum (AL) group. (A) Food intake of the four groups over the experimental period. The post-EODF group consumed ~78% as much as the ad libitum ...

At the time of the injury the animal body weights did not differ between the groups (Fig. 2B). Both the pre-EODF and the pair-fed calorie-restricted group started their restricted diet 3 weeks prior to injury, and we arranged the age of these animals in such a manner that all groups weighed the same at the time of injury. After injury all groups returned to pre-operative weight within 2 weeks, and continued to gain weight thereafter. However, both the pre-EODF and pair-fed restricted group gained less weight over time compared to the AL and post-EODF group. At the end of the experiment the three dietary-restricted groups weighed less than the AL group (post-EODF=−7%, pair-fed=−12.5%, pre-EODF=−16%; p<0.05).

Functional recovery after spinal cord injury

Horizontal ladder

On the horizontal ladder with irregular spacing there was a substantial increase in the percentage of hindlimb foot errors post-injury in all groups (Fig. 3; Metz and Whishaw, 2002). There were no statistically significant differences between groups for post-injury percentages of hindlimb footslips below the ladder rungs (Fig. 2; p<0.05). The rank order of the lowest to highest percentage of errors was consistent with the subsequent behavioral data, showing the two EODF groups at the top of the behavioral rankings; however, a Kruskal-Wallis rank order test did not reach statistically significant differences between groups.

FIG. 3.
Ladder crossing hindlimb footslip errors. There were no differences between the groups in the percentage of hindlimb footslip errors (footslips below the level of the rungs) in the post-injury period. All data are expressed as average±standard ...

CatWalk

One of the most-frequently used coordination parameter in the CatWalk is the RI, which indicates the regularity, or degree, to which the animal uses normal step sequence patterns (Deumens et al., 2006; Koopmans et al., 2005, 2009). During the post-injury period a two-way repeated-measures ANOVA revealed group differences (p<0.05; Fig. 4). Follow-up statistical analysis showed that the pre-EODF group had a higher RI than the ad libitum control group at 22 and 64 days post-injury, and both the ad libitum and pair-fed groups at 70 days post-injury (p<0.05 by one-way ANOVA; p<0.05 by Bonferroni post-hoc testing).

FIG. 4.
EODF improved CatWalk regularity index ratings. The regularity index from the CatWalk device gives a coordination score for each animal. While this percentage cannot be directly compared to the 95% coordination level used in the Basso-Beattie-Bresnahan ...

Other gait parameters altered as a result of compromised locomotion after injury include swing duration and stride length for both hindlimbs (HL) and forelimbs (FL). Typically, during coordinated walking, as observed in uninjured animals, the swing duration of the FL and HL are similar. After injury, swing duration timing becomes disrupted, with longer duration of the swing phase in the HL compared to the FL, resulting in negative values when you subtract the FL swing duration from the HL measurement (Fig. 5A). A two-way repeated-measures ANOVA revealed differences among the groups in the post-injury period (p<0.05). Both pre-EODF and post-EODF treatment resulted in a smaller difference between FL and HL swing duration scores compared to the ad libitum group at 50 and 64 days post-injury (p<0.05 by one-way ANOVA; p<0.05 by Bonferroni post-hoc testing). Both of the EODF groups' swing duration differences were not statistically significantly different than their pre-operative baseline levels at 50–64 days post-injury (p<0.05). The ad libitum and pair-fed groups retained a deficit throughout the testing period compared to their pre-operative baseline measurements (p<0.05).

A similar effect was observed for the difference between FL and HL stride lengths (Fig. 5B) during stepping. This difference is typically near zero before injury. After injury all groups showed large negative values, indicating relatively smaller FL strides compared to HL strides. A two-way repeated-measures ANOVA indicated group differences (p<0.05; Fig. 5B). At 64 and 70 days after injury the pre-EODF group showed improvement in recovery as evidenced by a smaller difference between FL – HL stride lengths compared to the ad libitum control group (p<0.05 by one-way ANOVA; p<0.05 by Bonferroni post-hoc testing; Fig. 5B).

Modified Basso-Beattie-Bresnahan open-field analysis

The BBB is the most widely used rating scale to evaluate locomotor function after a thoracic SCI in animal models (Basso et al., 1995, 1996). However, the most difficult and contentious component of the BBB is the accurate assessment of frequent (<95%) versus consistent (100%) coordination (Basso, 2004). Several researchers decided to use footprint analysis to assist in the measurement of coordination (Deumens et al., 2006; Koopmans et al., 2005, 2009). Therefore, we used the catwalk RI as an objective measurement of coordination and incorporated it into our weekly BBB measurements (Fig. 6). We designated this measurement as a regularity indexed BBB score (RI-BBB). There was a drop in RI-BBB after injury, which improved over time (p<0.05 by two-way repeated-measures ANOVA; Fig. 6). Analysis over this same time period also indicated a significant effect of group assignment (p<0.05 by two-way repeated-measures ANOVA). The pre-EODF group (at 50, 64, and 70 days post-injury), and the post-EODF group (at 64 and 70 days post-injury) achieved significantly higher RI-BBB scores than both the ad libitum control and the pair-fed groups (p<0.05 by one-way ANOVA; p<0.05 by Bonferroni post-hoc testing). The ad libitum control and pair-fed groups reached an average score of 12.42±0.69 and 12.17±0.31, respectively, indicating plantar stepping with frequent to consistent weight bearing and occasional FL-HL coordination. In contrast, the pre-EODF and post-EODF animals showed plantar stepping with consistent weight support, and consistent FL-HL coordination (14.17±0.75 and 14.77±0.58, respectively).

FIG. 6.
EODF improved the regularity indexed BBB (RI-BBB). Using the RI to measure the degree of coordination, we observed a significant effect of group assignment (p<0.05 by two-way repeated-measures ANOVA). The pre-EODF group (at 50, 64, and 70 days ...

Lesion size and tissue sparing

With our previous finding of a reduced lesion size after a cervical spinal cord injury in the EODF group, we wanted to ascertain if we would see similar results in our thoracic injury model (Plunet et al., 2008, 2010). The spared white matter rim was quantified in cross-sections at 400-μm intervals spanning from 2800 μm rostral to 2800 μm caudal to the epicenter. As expected, the contusion injury to the thoracic spinal cord resulted in a dramatic loss of gray and white matter at the epicenter, with only a thin rim of residual white matter (Fig. 7A). In all four groups the lesion area extended both rostrally and caudally for 1600–2000 μm. No group differences in the amount of spared white matter were observed (p<0.05 by two-way repeated-measures ANOVA).

FIG. 7.
Spared white and gray matter. (A) The rostral and caudal extent of spared white matter was quantified at 400-μm intervals spanning from 2800 μm rostral to 2800 μm caudal to the epicenter. No group differences in ...

Gray matter was virtually eliminated at the epicenter of the injury, but further rostral and caudal to the injury there was some spared gray matter in all groups (Fig. 7B). No group differences were found between the groups in the amount of spared gray matter (p>0.05 by two-way repeated-measures ANOVA), although the amount of sparing 2000 μm rostral to the lesion site correlated with behavioral outcomes (see correlation section below).

Serotonergic fibers

With EODF reported to increase plasticity (Fontan-Lozano et al., 2007; Plunet et al., 2008), we wanted to examine if increased sprouting could account for the functional differences between the groups we observed after SCI. The delayed functional recovery would also be more consistent with enhanced plasticity rather than neuroprotection. Detection of descending raphe-spinal serotonergic tracts was visualized by serotonin transporter (SERT) immunofluorescence. 5-HT transporter (SERT) immunostaining has a better correlation with return of function than 5-HT, and hence we used SERT immunostaining to assess serotonin's influence on functional recovery in our experiment (Saruhashi et al., 2009). 5-HT fibers at the L1–L2 level (below the level of injury) are known to be important for interlimb coordination (Jordan et al., 2008; Schmidt and Jordan, 2000).

Caudal to the injury we found no statistically significant differences among the groups in SERT immunostaining density in the ventral gray matter horns (p=0.14 by one-way repeated-measures ANOVA; Fig. 8), although there was a trend toward higher SERT staining densities in both EODF groups. We additionally examined the more specific location of motor neurons in lamina IX (Fig. 9A), but again the trend toward higher values in the post-EODF group did not reach statistical significance (p>0.05 by one-way ANOVA). Since some of our behavioral differences among groups centered around interlimb coordination, and 5-HT fibers at the L1–L2 level (below the level of injury) are known to be important for interlimb coordination, we made a more detailed examination (Jordan et al., 2008; Schmidt and Jordan, 2000). The physiological functioning of 5-HT requires some threshold amount on each side of the spinal cord (Saruhashi et al., 1996, 2009). Hence, we obtained a symmetry score by taking the ratio of SERT immunostaining in lamina IX on one side compared to the other side. While a conservative one-way ANOVA did not show a difference among the groups (Fig. 9B), a Pearson correlation analysis revealed interesting relationships between histology and behavior, as did a number of the SERT parameters we measured.

FIG. 8.
Serotonin transporter (SERT) sparing/sprouting caudal to the injury in the ventral gray matter. Caudal to the injury we found no statistically significant differences among the groups for SERT immunostaining density in the ventral gray matter (p=0.14 ...
FIG. 9.
Serotonin transporter (SERT) expression in Rexed lamina IX caudal to injury and symmetry scores. (A) A one-way ANOVA found no differences between groups in the amount of SERT immunostaining caudal to the injury in lamina IX (p>0.05). (B) The level ...

Correlation of behavioral outcomes and histology with behavior

While we did not observe significant group differences on any of the histological parameters, we explored if any of these parameters correlated with the various functional outcomes using Pearson correlation analyses, and by examining correlations between the various behaviors (Tables 1 and and22).

Table 1.
Strong Correlations between Different Functional Recovery Behaviors
Table 2.
Histological Outcomes Were Significantly Correlated with Behavioral Measurements

First we analyzed the relationships between various behavioral outcomes (i.e., between each other; Table 1). We found a high degree of significant correlations between all the behaviors, with the exception of percentage of ladder errors, which did not correlate with stride length differences (Table 1). Interestingly, the percentage of ladder errors (which did not differ between groups) correlated with all the other behaviors, other than the one mentioned exception (stride length).

We then examined if any of our histological measurements correlated with the various behavioral outcomes. Overall white matter sparing did not correlate with behavior, or any of the individual sparing measurements at the epicenter, or those made at various distances rostral or caudal to the injury site (p<0.05 by Pearson correlation). Overall gray matter sparing did not significantly correlate with any of the behaviors, but the amount of gray matter spared 2000 μm rostral to the lesion did significantly correlate with two of the five tested behaviors (p<0.05; Table 2).

Despite the absence of significant group differences among the various SERT immunostaining measurements, we observed correlations with several behavioral outcomes. Both the ventral gray matter (Fig. 8) and the lamina IX (Fig. 9A) caudal to the injury site significantly correlated with several behavioral measurements (p<0.05; Table 2). Lamina IX SERT immunostaining levels correlated with swing duration differences and percentage of ladder errors, while the broader ventral horn gray matter SERT staining correlated with four out of the five behaviors (swing duration differences, percentage of ladder errors, RI-BBB, and the RI). Finally, the symmetrical level of SERT immunostaining in lamina IX (Fig. 9B) significantly correlated with RI, swing duration differences, and percentage of ladder errors.

In addition, we examined if correlations of the histology/behavior relationships existed independently in each of the four different animal groups, and for each possible histology/behavior interaction. Of the 80 possible correlations, only 5 reached significance. In the control group there was a correlation between gray matter sparing 2000 μm caudal to the injury site and swing duration differences, as well as with the RI-BBB; and the RI correlated with the caudal lamina IX SERT symmetry score. The only other two significant correlations were found in the post-EODF group between the caudal lamina IX SERT levels and ladder errors, and between the caudal ventral gray matter SERT levels and swing duration differences. There were no significant relationships between behavior and histology for the other two groups.

Discussion

Here we report that EODF improves functional recovery after a thoracic spinal cord contusion injury of moderate severity in rats. Both EODF starting 3 weeks before the injury, or more importantly, starting it after the injury, were effective. The pre-EODF and post-EODF groups reached significantly higher scores on the RI-BBB for open-field locomotion when compared to the ad libitum control group, but also the pair-fed group. The pair-fed group consumed the same total amount of calories offered daily. In addition, on several parameters measured with the CatWalk gait analysis device, both EODF feeding regimens elicited improved functional recovery. Hence, EODF has prophylactic and therapeutic potential after thoracic contusion SCI, and these findings are consistent with our previous findings with pre-EODF and post-EODF in a cervical SCI model, the dorsolateral funiculus crush (Plunet et al., 2008, 2010).

While both EODF groups had better functional recovery than the two control groups (ad libitum and pair-fed), the pre-EODF group (starting the diet 3 weeks prior to injury) improved functional recovery to a greater extent than the post-EODF group (Table 3). This observation is consistent with our two separate EODF studies after cervical SCI, although they were not performed at the same time and therefore cannot be directly compared (Plunet et al., 2008, 2010). One can speculate that cellular pathways for neuroprotection have been already activated by 3 weeks of pre-EODF, and that this provides benefits during the early secondary injury phase. In contrast, this activation of EODF-induced effects may occur too slowly to influence the early cascades of secondary death and inflammation if the diet is started after injury. The clinical significance of our pre-EODF data is that such a regimen could conceivably be implemented prior to elective interventions that put the spinal cord at risk, such as surgery for deformities or tumors. Additionally, EODF started after injury, which would have wider clinical applications, also improved functional recovery after SCI.

Table 3.
Summary of Behavioral Effects Observed with Different Dietary Strategies after Thoracic Contusion Injury Compared to the ad Libitum Control Group

Daily caloric restriction (pair-fed group), in contrast to EODF, did not improve functional outcomes after thoracic contusion injury, and the pair-fed rats showed similar behavioral performance as the ad libitum group. This differential outcome between the CR and EODF groups is consistent with previous research in mice showing increased neuroprotective efficacy of pre-EODF compared to caloric restriction after kainate-induced brain injury (Anson et al., 2003). This suggests that similar levels of calorie restriction do not produce the same behavioral outcome, and the specific timing of food/fasting can make a substantial difference in the effects. Anson and coworkers suggested that differences in trophic factors may play a role. It is unknown how a change in metabolism and gene expression elicited by 24 h of fasting, followed by a feasting and free access to food for 24 h, contributes to the increased functional recovery seen after SCI. We propose that the 24-h fasting period increases blood ketone levels, which could exert neuroprotective effects. Such an increase in ketones is not seen on a caloric-restriction regimen (Anson et al., 2003), such as the pair-fed control feeding regimen. This hypothesis is supported by recent data from our laboratory revealing that a ketogenic diet, which also increases ketone levels, after cervical hemi-contusion injuries in rats elicits robust improvements in behavioral outcomes (Streijger et al., in submission). In addition, it is conceivable that 24 h of feasting (eating ~140–160% of normal intake) contributes to the functional recovery by altering fundamental metabolic pathways, such as stimulation of the mTOR pathway, as has recently been reported in the brains of EODF animals, but not in similarly caloric-restricted animals that were fed every day (Martin et al., 2008). Increased levels of mTOR have recently been shown to enhance the regenerative capacity of corticospinal axons (Liu et al., 2010). Further research is necessary to clarify the relative contributions of these possible fasting and/or feasting effects.

In light of our previous findings of a reduced lesion size after a cervical SCI in both pre-EODF and post-EODF groups (Plunet et al., 2008, 2010), we examined here if similar neuroprotection is seen in our thoracic injury model. Despite functional improvements in both EODF groups after thoracic SCI, we did not observe any differences in the amount of spared white or gray matter between the groups. Of note, in our cervical model EODF rescued mainly gray matter, and gray matter is typically entirely destroyed after thoracic contusions. However, there was a trend toward improved gray matter rescue at a distance of 2000–1600 μm from the epicenter. It is conceivable that the more complete and larger injury seen after thoracic contusion, compared to the unilateral cervical crush injury, masks any of the differences in neuroprotection; in other words, an equally small rim of rescued tissue would be more evident and easier to detect in a small lesion. Hence a small neuroprotective effect of EODF might have gone undetected in this more severe thoracic contusion model.

EODF is known to alter the expression of a large number of proteins that could account for increased plasticity (Mattson et al., 2003), and indeed EODF increased axonal plasticity as measured by long-term changes in synaptic efficiency, coinciding with increases in NMDA receptor subunit NR2B, and sprouting of spared corticospinal axons (Fontan-Lozano et al., 2007; Plunet et al., 2008). The delayed functional recovery (50 days) in the both EODF groups would be consistent with enhanced neural plasticity being the major mechanistic player. In the cervical injury model we observed increased sprouting of the intact corticospinal tract (Plunet et al., 2008). In the present study we quantified serotonergic axons, as this system plays an important role in limb coordination (Jordan et al., 2008; Schmidt and Jordan, 2000), and hence is often studied in thoracic SCI models. While the several parameters of SERT sprouting data correlated with functional recovery, we did not detect group differences among our multiple groups using conservative ANOVA statistics. These results suggest that additional fiber systems might be playing a role, and that individual fiber systems may only show subtle changes.

These results also raise the possibility that changes at the cortical level could have occurred that contributed to the change in functional recovery observed in the EODF groups. The potential cortical level changes could have occurred at a structural level and/or at a molecular level. Since EODF can have systemic affects, including in the brain, this could provide additional mechanisms for beneficial outcomes. Both calorie restriction and EODF have been reported to improve learning/memory in older animals, though there are other researchers that found no improvement (Bond et al., 1989; Beatty et al., 1987; Idrobo et al., 1987; Pitsikas and Algeri, 1992). Improved learning/memory in younger animals, such as those used in our experiment, is not established. In our study we did not specifically examine any cortical structural plasticity changes induced by EODF that could account for our behavioral results. An additional possibility is that a cortical learning effect may account for some of these improvements. However, we think that this is unlikely for several reasons. The behavior with the highest potential for learning, the skilled ladder crossing test, revealed no differences among the groups. The other two tests, open-field, and CatWalk, involve arguably less skilled locomotor movements (i.e., walking/running). While there is likely a component of re-learning after SCI for these motor patterns, these are more likely “hard wired” in comparison to a more complex task such as crossing an irregular ladder with specific foot placements on the slim rungs. Additionally, the equally calorie-restricted pair-fed group did not show any improvement in motor recovery after SCI, and if memory/learning was playing a role we would have expected similar improvements in motor recovery in this group, since the level of calorie restriction these animals were on is similar to what is reported to improve memory/learning, at least in older animals. Overall, this suggests that improved memory/learning did not play a major role in the motor recovery seen after SCI in this experiment.

Conclusion

Here we provide further evidence that rats maintained on an EODF diet after SCI show improved motor function. Both the prophylactic pre-EODF and therapeutic post-injury-initiated EODF resulted in improved functional recovery in a thoracic injury contusion model compared to the control ad libitum and pair-fed calorie-restricted groups. Additionally, the pre-EODF performed slightly better than the post-EODF group. Finally, daily calorie restriction at the same level as the EODF groups did not appear to produce the same positive effects as EODF on behavioral recovery after SCI. However, we could not detect significant neuroprotective effects of EODF in this injury model, nor were there any pronounced group differences in SERT sparing/sprouting, despite a significant correlation of SERT staining with behavioral outcomes. It is conceivable that multiple mechanisms are mediating the improvements seen in functional recovery, which when measured in isolation may not reach significance. The main result of improved functional recovery elicited by this safe, simple, and inexpensive EODF paradigm in two different experimental groups of thoracic contusion adds to our previous findings of improved recovery after cervical SCI. Taken together, this body of data supports the notion that specific dietary strategies might eventually benefit acutely injured patients with SCI; however, more pre-clinical data about the possible mechanisms and side effects of this approach are needed.

Acknowledgments

This work was supported by a grant from the Canadian Institute for Health Research (CIHR) to W.T.

Author Disclosure Statement

No competing financial interests exist.

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