Attenuating Experimental Spinal Cord Injury by Hyperbaric Oxygen: Stimulating Production of Vasculoendothelial and Glial Cell Line-Derived Neurotrophic Growth Factors and Interleukin-10

Abstract

The present study was carried out to further examine the mechanisms underlying the beneficial effects of hyperbaric oxygen (HBO₂) on experimental spinal cord injury (SCI). Rats were divided into three major groups: (1) sham operation (laminectomy only); (2) laminectomy + SCI + normobaric air (NBA; 21% oxygen at 1 ATA); and (3) laminectomy + SCI + HBO₂ (100% oxygen at 2.5 ATA for 2 h). Spinal cord injury was induced by compressing the spinal cord for 1 min with an aneurysm clip calibrated to a closing pressure of 55 g. HBO₂ therapy was begun immediately after SCI. Behavioral tests of hindlimb motor function as measured by the Basso, Beattie, and Bresnahan (BBB) locomotor scale was conducted on days 1–7 post-SCI. The triphenyltetrazolium chloride staining assay and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate biotin nick-end labeling assay were also conducted after SCI to evaluate spinal cord infarction and apoptosis, respectively. Cells positive for glial cell line–derived neurotrophic nerve growth factor (GDNF) and vascular endothelial growth factor (VEGF) and cytokines in the injured spinal cord were assayed by immunofluorescence and commercial kits, respectively. It was found that HBO₂ therapy significantly attenuated SCI-induced hindlimb dysfunction, and spinal cord infarction and apoptosis, as well as overproduction of spinal cord interleukin-1β and tumor necrosis factor-α. In contrast, the numbers of both GDNF-positive and VEGF-positive cells and production of spinal cord interleukin-10 after SCI were all significantly increased by HBO₂. These data suggest that HBO₂ may attenuate experimental SCI by stimulating production of GDNF, VEGF, and interleukin-10.

Introduction

H yperbaric oxygen (HBO₂) therapy for patients with stroke, atherosclerosis, cerebral palsy, intracranial hypertension, headache, and brain and spinal cord injury (SCI) appears promising and warrants further investigation (Al Waili et al., 2005). HBO₂ has been reported to accelerate neurologic recovery after SCI by ameliorating mitochondrial dysfunction in the motor cortex and spinal cord, arresting the spread of hemorrhage, reversing hypoxia, and reducing edema (Al Waili et al., 2005).

In an experimental model of SCI, the end-points of the inflammatory response have been determined: (1) histological damage; (2) motor dysfunction; (3) neutrophil infiltration; (4) cytokine expression (tumor necrosis factor-α and interleukin-1β); and (5) apoptosis (TUNEL staining; Genovese et al., 2006). In addition, it was shown that human umbilical cord blood–derived CD34⁺ cells may attenuate SCI by stimulating vascular endothelial growth factor (VEGF) and glial cell line–derived neurotrophic nerve growth factor (GDNF) production (Kao et al., 2008a). These observations raise the possibility that HBO₂ may induce improved outcomes after SCI by inhibiting the above-mentioned end-points of the inflammatory response, and by stimulating production of both VEGF and GDNF. To explore this hypothesis, in the present study the temporal profiles of histological damage, motor dysfunction, neutrophil infiltration, proinflammatory cytokine overproduction, apoptosis, and production of both VEGF and GDNF after SCI were assessed in rats with or without HBO₂ therapy.

Methods

Inducing spinal cord ischemia

Adult male Sprague-Dawley rats (weighing 256 ± 11 g) were obtained from the Animal Resource Center of the National Science Council of the Republic of China (Taipei, Taiwan). The animals were housed in groups of four at an ambient temperature of 22 ± 1°C with a 12-h light-dark cycle. Pellet rat chow and tap water were available ad libitum. All protocols were approved by the Animal Ethics Committee of the Chi Mei Medical Center (Tainan, Taiwan), in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and the Guidelines of the Animal Welfare Act. A laminectomy with vertebral peduncle removal was performed at T8 or T9 on rats anesthetized with sodium pentobarbital (25 mg/kg IP; Sigma Chemical Co., St. Louis, MO), and a mixture containing ketamine (44 mg/kg IM; Nankang Pharmaceutical Co., Taipei, Taiwan), atropine (0.02633 mg/kg IM; Sintong Chemical Industrial Co, Taoyuan, Taiwan), and xylazine (6.77 mg/kg IM; Bayer, Leuerkusen, Germany). The jaws of a calibrated aneurysm clip with a closing pressure of 55 g were placed on the dorsal and ventral surfaces of the spinal cord and left in place for 1 min (Takahashi et al., 2003). The sham-operated control animals received the same laminectomy, but did not undergo compression. All animals were given 0.1 mL daily of Baytril (Bayer) antibiotic for 3 days postoperatively. Animals with SCI were individually housed on special bedding to prevent skin breakdown, and their bowels and bladders were manually expressed twice daily. Food and water were freely accessible at a lowered height in their cages.

Hyperbaric oxygen therapy and animal groups

Animals were assigned randomly to one of the following three groups. In the sham-operated group, immediately after surgery they were exposed to normobaric air (NBA; 21% oxygen at 1 ATA). In the hyperbaric oxygen (HBO₂)-treated group, immediately after surgery they were treated with HBO₂ (Po ₂ = 100% at 2.5 ATA) for 2 h. The chamber filled with pure oxygen was pressurized to 2.5 ATA at a rate of 7 pounds per square inch/min for 2 h and was terminated at the decompression rate of 2.9 pounds per square-inch/min. The experimental plan is summarized in Table 1.

Table 1.

Experimental Design

Group	BBB scale	TTC staining	TUNEL staining	VEGF staining	GDNF staining	MPO + cytokine assay
1. Sham-operated (SO) rats	1 day after SO (n = 8)	1 day after SO (n = 8)	1 day after SCI (n = 8)	1 day after SO (n = 8)	1 day after SO (n = 8)	4 hours after SO (n = 8)
	4 day after SO (n = 8)	4 day after SO (n = 8)	4 day after SCI (n = 8)	4 day after SO (n = 8)	4 day after SO (n = 8)
	7 day after SO (n = 8)	7 day after SO (n = 8)	7 day after SCI (n = 8)	7 day after SO (n = 8)	7 day after SO (n = 8)
	30 day after SO (n = 8)	30 day after SO (n = 8)	30 day after SCI(n = 8)	30 day after SO (n = 8)	30 day after SO (n = 8)
2. NBA-treated SCI-operated rats	1 day after SCI (n = 8)	1 day after SCI (n = 8)	1 day after SCI (n = 8)	1 day after SCI (n = 8)	1 day after SCI (n = 8)	4 hours after SCI (n = 8)
	4 day after SCI (n = 8)	4 day after SCI (n = 8)	4 day after SCI (n = 8)	4 day after SCI (n = 8)	4 day after SCI (n = 8)
	7 day after SCI (n = 8)	7 day after SCI (n = 8)	7 day after SCI (n = 8)	7 day after SCI (n = 8)	7 day after SCI (n = 8)
	30 day after SCI (n = 8)	30 day after SCI (n = 8)	30 day after SCI (n = 8)	30 day after SCI (n = 8)	30 day after SCI (n = 8)
3. HBO₂-treated SCI-operated rats	1 day after SCI (n = 8)	1 day after SCI (n = 8)	1 day after SCI (n = 8)	1 day after SCI (n = 8)	1 day after SCI (n = 8)	4 hours after SCI (n = 8)
	4 day after SCI (n = 8)	4 day after SCI (n = 8)	4 day after SCI (n = 8)	4 day after SCI (n = 8)	4 day after SCI (n = 8)
	7 day after SCI (n = 8)	7 day after SCI (n = 8)	7 day after SCI (n = 8)	7 day after SCI (n = 8)	7 day after SCI (n = 8)
	30 day after SCI (n = 8)	30 day after SCI (n = 8)	30 day after SCI (n = 8)	30 day after SCI (n = 8)	30 day after SCI (n = 8)

BBB, Basso, Beattie, and Bresnahan scale; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate biotin nick-end labeling; TTC, triphenyltetrazolium chloride; VEGF, vascular endothelial growth factor; GDNF, glial cell line–derived neurotrophic nerve growth factor; HBO₂, hyperbaric oxygen; MPO, myeloperoxidase; NBA, normobaric air; SCI, spinal cord injury.

In Experiment 1, SCI was performed randomly in rats treated with NBA (n = 8) or HBO₂ (n = 8), and their effects on the Basso, Beattie, and Bresnahan (BBB) locomotor scores and spinal cord infarct zones were assessed on days 1–7 after SCI. The other eight sham-operated rats were used as controls.

In Experiment 2, SCI was performed randomly in rats treated with NBA (n = 8) or HBO₂ (n = 8), and their effects on the levels of myeloperoxidase (MPO) activity, and on levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-10 (IL-10) in the injured spinal cord were assessed 4 h after SCI. The other eight sham-operated rats were used as controls.

In Experiment 3, SCI was randomly performed in rats treated with NBA (n = 8) or HBO₂ (n = 8), and their effects on the amounts of vascular endothelial growth factor (VEGF)-positive cells, glial cell line–derived neurotrophic nerve growth factor (GDNF)-positive cells, and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL)-positive cells in the injured spinal cord were assessed 1, 4, or 7 days after SCI. The other eight sham-operated rats were used as controls.

Assessing hindlimb locomotor function

A behavioral test was performed to measure the functional recovery of the rat's hindlimb according to the procedure described by Basso and associates (1995). The scale used to assess hindlimb function with this procedure ranges from a score of 0, indicating no spontaneous movement, to a maximum score of 21, with increasing score indicating the use of individual joints, coordinated joint movement, coordinated limb movement, weight-bearing, and other types of function. The rats were first gently adapted to the open field used for the test. After a rat had walked in the open field, two investigators conducted 4-min testing sessions on each leg. Behavioral outcomes and examples of specific BBB locomotor scores were recorded using a digital video recorder.

Assessing spinal cord infarction volume

The triphenyltetrazolium chloride (TTC) staining procedure is described in detail elsewhere (Wang et al., 1997). All animals were killed at days 1, 4, and 7 after SCI with deep anesthesia (100 mg/kg sodium pentobarbital IP). The spinal cord tissue was then removed, immersed in cold saline for 5 min, and sliced into 2.0-mm sections with a tissue slicer. The spinal cord slices were incubated in 2% TTC dissolved in PBS for 30 min at 37°C, and then transferred to 5% formaldehyde for fixation. The lesion sites were evaluated blindly by serial reconstructions. The infarction volume, as revealed by pale TTC staining (whitish color) indicating dehydrogenase-deficient tissue, was measured in each slice and summed using computerized planimetry (PC-Based Image Tools Software; Media Cybernetics, Inc., Bethesda, MD). The infarction volume was calculated as 2 mm (thickness of the slice) × (sum of the infarction area in all spinal cord slices [mm²]; Wang et al., 1997). The histological analyses were performed blindly.

Cytokine analysis

For the determination of IL-1β, TNF-α, and IL-10 in the injured spinal cord, samples were taken 1, 4, or 7 days after SCI, or the equivalent time for the sham-operated rats. The concentrations of these cytokines in the injured spinal cord were determined using double-antibody sandwich ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. The optical densities were read on a plate reader set at 450 nm for IL-1β, TNF-α, and IL-10. The concentrations of IL-1β, TNF-α, and IL-10 in the samples were calculated from a standard curve multiplied by the dilution factor and is expressed in picograms per gram.

Assessing spinal cord apoptosis

The TUNEL assay used the same spinal cord tissues used for histological verification. The color was developed using 3,3-diamino-benzidine tetrachloride (DAB). The sections were treated thusly: (1) xylene- and ethanol-treated for paraffin removal and for dehydration before washing with PBS and incubation in 3% H₂O₂ solution for 20 min; (2) treated with 5 μg/mL proteinase k for 2 min at room temperature, and re-washing with 0.1 M PBS (pH 7.4), followed by the TUNEL-reaction mixture (terminal deoxynucleotidyl transferase, nucleotide; Roche, Mannheim, Germany) at 37°C for 1 h, and afterward washing with distilled water; (3) re-incubated in an anti-fluorescein antibody-conjugated with horseradish peroxidase at room temperature for 30 min, and re-washing before visualization using the avidin-biotin-peroxidase complex (ABC) technique and 0.05% (Sigma Chemical) as a chromogen. The numbers of TUNEL-positive cells were counted by a pathologist in 30 fields per section (×200 magnification) blinded to study group.

Determining myeloperoxidase activity

MPO activity, an indicator of polymorphonuclear leukocyte accumulation, was determined in the spinal cord tissues as previously described (Mullane et al., 1985) at 4 h post-SCI. This time point was chosen to be in agreement with another study (Hamada et al., 1996). MPO activity was defined as the quantity of enzyme degrading 1 μmol of peroxide min⁻¹ at 37°C, and is expressed in milliunit grams⁻¹ of wet tissue.

Assessing both VEGF-positive and GDNF-positive cells

Autofluorescence was first quenched using the method of Vendrame and colleagues (2004), after which the spinal cord sections were incubated with PBS containing anti-VEGF or anti-GDNF mouse antibody in 1:200 dilution; then the sections were detected with Alexa-Fluor 568 goat anti-mouse (IgG) antibody. The slides were examined under epifluorescence on an Olympus BX60 microscope (Olympus, Center Valley, PA).

Statistical analysis

The baseline BBB scale scores, spinal cord infarction zones, and numbers of TUNEL-positive cells, VEGF-positive cells, and GDNF-positive cells, and concentrations of IL-1β, IL-10, TNF-α, and MPO in the three groups were compared with the use of repeated-measures analysis of variance (ANOVA). This was conducted to test the treatment-by-time interactions, and the effect of treatment over time on each score. The Duncan's multiple-range test was used for post-hoc multiple comparisons among means. Data are presented as the mean ± SEM, and statistical significance was set at p < 0.05.

Results

Basso, Beattie, and Bresnahan locomotor scale, triphenyltetrazolium chloride stain, and apoptotic cell count

Behavioral tests of hindlimb motor function were conducted on days 1, 4, and 7 after SCI to determine whether HBO₂ therapy adopted immediately after SCI would produce beneficial effects. In Figure 1, we see that although NBA treatment was slightly effective as assessed by the BBB locomotor scale, the SCI-induced hindlimb motor deficits were greatly ameliorated by HBO₂ therapy at days 4–7 after SCI. At 1 month after SCI, HBO₂ therapy-improved motor performance was sustained (Fig. 1). Immediately after motor performance assessment on days 1, 4, and 7 after SCI, the animals were killed for TTC staining. NBA-treated SCI animals showed severe infarctions, characterized by pale TTC staining (whitish color) in the white matter of all injured spinal cord sections. A typical example is shown in the top panels of Figure 2, which also show that HBO₂, but not NBA, significantly limited the spinal cord infarct at 4–7 days after SCI. In addition, on days 4–7 after SCI, TUNEL staining revealed that spinal cord apoptosis (as evidenced by an increase in TUNEL-positive cells in the injured spinal cord) was significantly reduced by HBO₂ therapy adopted immediately after SCI (Fig. 3).

FIG. 1.

Effects of hyperbaric oxygen (HBO₂) on hindlimb motor disturbances after spinal cord injury. The degree of motor disturbances was assessed daily until 30 days post-SCI by the Basso, Beattie, and Bresnahan (BBB) criteria. After injury, normobaric air (NBA)-treated SCI rats () had significant deficits in hindlimb movement (*p < 0.01) compared with sham-operated rats (□). Treatment with HBO₂ () reduced motor disturbances seen after SCI (^† p < 0.01). Values are mean ± standard error of the mean of 8 rats for each group.

FIG. 2.

Effects of hyperbaric oxygen (HBO₂) on the injured area. After injury, the infarction zone in the spinal cord of normobaric air (NBA)-treated spinal cord injured (SCI) rats () was significantly increased 1–7 days after injury compared with sham-injured rats (□). Treatment with HBO₂ () significantly reduced the SCI-induced increase in the infarction zone. Data are means ± standard error of the mean of 8 rats for each group (*p < 0.01 versus sham animals; ^† p < 0.01 versus SCI).

FIG. 3.

Effects of hyperbaric oxygen (HBO₂) on numbers of TUNEL-positive cells. After injury, the numbers of TUNEL-positive cells in the spinal cords of normobaric air (NBA)-treated SCI-operated rats () were significantly increased at 4–7 days after injury compared with sham rats (□). Treatment with HBO₂ () significantly reduced the SCI-induced increase in TUNEL-positive cells. Data are means ± standard error of the mean of 8 rats for each group (*p < 0.01 versus sham animals; ^† p < 0.01 versus SCI; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate biotin nick-end labeling assay; SCI, spinal cord injury).

GDNF-positive and VEGF-positive cell counts

To elucidate whether VEGF or GDNF could be induced in the spinal cord-injured area by HBO₂, analysis of the numbers of VEGF-positive cells and GDNF-positive cells in the spinal cord was performed, which revealed that spinal cord-injured areas at 4–7 days after HBO₂ therapy displayed more VEGF-positive cells (Fig. 4) and GDNF-positive cells (Fig. 5) than those in the NBA-treated SCI animals.

FIG. 4.

Effects of hyperbaric oxygen (HBO₂) on the numbers of vascular endothelial growth factor (VEGF)-positive cells. After injury, the numbers of VEGF-positive cells in the spinal cord of normobaric air (NBA)-treated SCI rats () were slightly increased at 4–7 days after injury compared with sham rats (□). Treatment with HBO () significantly enhanced the SCI-induced increase in VEGF-positive cells. Data are means ± standard error of the mean of 8 rats for each group (*p < 0.05 versus sham animals; ^† p < 0.01 versus SCI + NBA animals; SCI, spinal cord injury).

FIG. 5.

Effects of hyperbaric oxygen (HBO₂) on numbers of glial cell line–derived neurotrophic nerve growth factor (GDNF)-positive cells. After injury, the numbers of GDNF-positive cells in the spinal cord of normobaric air (NBA)-treated SCI rats () were slightly increased at 4–7 days after injury compared with sham rats (□). Treatment with HBO () significantly enhanced the SCI-induced increase in the numbers of GDNF-positive cells. Data are means ± standard error of the mean of 8 rats for each group (*p < 0.05 versus sham animals; ^† p < 0.01 versus SCI + NBA animals; SCI, spinal cord injury).

Proinflammatory and anti-inflammatory cytokines

To elucidate whether production of proinflammatory cytokines (IL-1β and TNF-α), anti-inflammatory cytokines (e.g., IL-10), and an indicator of polymorphonuclear leukocyte accumulation (e.g., MPO) were affected by HBO in spinal cord injury, biochemical analysis was performed 4 h after injury. It was found that compared with NBA-treated SCI rats, HBO₂-treated SCI rats had less production of IL-1β, TNF-α, and MPO, but more production of IL-10, in the injured spinal cord (Fig. 6).

FIG. 6.

Effects of hyperbaric oxygen (HBO₂) on myeloperoxidase (MPO) activity and levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-10. After injury, MPO activity and levels of TNF-α and IL-1β in the spinal cords of normobaric air (NBA)-treated SCI rats (▪) was significantly increased at 4 h after injury compared with sham rats (□). As compared with NBA-treated sham-operated or NBA-treated SCI rats, the HBO₂-treated sham-operated and HBO₂-treated SCI rats had significantly higher levels of IL-10 at 4 h after surgery. Treatment with HBO₂ () significantly reduced the SCI-induced increase in MPO, TNF-α, and IL-1β activity. Data are means ± standard error of the mean of 8 rats for each group (*p < 0.05 versus sham animals; ^† p < 0.01 versus NBA + SCI animals).

Discussion

In this study, spinal cord injury in rats resulted in severe trauma characterized by spinal cord infarction, neutrophil infiltration, and production of proinflammatory cytokines (e.g., IL-1β and TNF-α), that caused tissue damage, apoptosis, and hindlimb locomotor dysfunction. Treating the SCI rats with HBO₂ significantly reduced: (1) the degree of spinal cord inflammation and infarction; (2) neutrophil infiltration; (3) proinflammatory cytokine production; (4) apoptosis; and (5) hindlimb locomotor dysfunction. Also, treating the SCI rats with HBO₂ significantly increased the amount of expression of both VEGF and GDNF, as well as production of the anti-inflammatory cytokine interleukin-10. Taken together, our results clearly demonstrate that treatment with HBO₂ reduced the development of SCI-associated inflammation and attenuated tissue injury.

It has been shown that after experimental SCI, the levels of both TNF-α and IL-1β are significantly increased in the injured spinal cord within the first few hours post-injury (Genovese et al., 2006; Harrington et al., 2005; Hayashi et al., 2000). Moreover, in SCI, the expression of proinflammatory cytokines, including TNF-α and IL-1β, at the site of injury affects subsequent events occurring after SCI (Streit et al., 1998). There is also evidence that TNF-α and IL-1β play an important role in the induction of inducible nitric oxide synthase (iNOS; Matsuyama et al., 1998), cyclooxygenase-2 (COX-2; Genovese et al., 2006; Sairanen et al., 1998), and reactive oxygen species (ROS; Xu et al., 2001), which are known to be responsible for the secondary neuronal damage seen post-SCI. In particular, it was shown that TNF-α induced apoptosis in oligodendrocytes both in vitro and in vivo (Muzio et al., 1997), via the activation of caspase-3 and caspase-8 (Hisahara et al., 1997). In addition, IL-1β acts as an extracellular signal that initiates apoptosis in neurons and oligodendrocytes after SCI (Ehrlich et al., 1999; Fankhauser et al., 2000; Takahashi et al., 2003). Intrathecal administration of an IL-1 receptor antagonist (Wang et al., 2005), or intraperitoneal administration of etanercept (a TNF-α antagonist) (Genovese et al., 2006), significantly reduced the numbers of TUNEL-positive cells in the injured spinal cord in rats. The current study further shows that HBO₂ ameliorates spinal cord inflammation after SCI by decreasing the production of both TNF-α and IL-1β, but increasing IL-10 production. It is believed that IL-10 has important anti-inflammatory properties through suppression of TNF-α, IL-1β, and other proinflammatory cytokines (Oberholzer et al., 2000). In fact, endotoxin-induced inflammation was shown to impair neurogenesis (Ekdahl et al., 2003), whereas blockade of inflammation was shown to restore neurogenesis (Hoehn et al., 2005; Valable et al., 2003; Monje et al., 2003).

VEGF was shown to act on the vasculature, having an important role in providing an environment conductive to neurogenesis (Taguchi et al., 2004; Valable et al., 2003), whereas GDNF promotes axonal growth and cellular protection in injured motor neurons (Blesch and Tuszynski, 2001). Human umbilical cord blood-derived CD34⁺ cells restored normal hindlimb locomotion in an SCI rat model by stimulating production of both VEGF and GDNF in the injured spinal cord (Kao et al., 2008a). Direct delivery of GDNF also improved outcomes of post-SCI by attenuating spinal cord infarction and apoptosis (Kao et al., 2008b). Like human umbilical cord blood-derived CD34⁺ cells, HBO₂ therapy was shown to induce the genetic expression of GDNF and reduce apoptosis in spinal cord injury (Yu et al., 2004). Our results further demonstrate that HBO₂ may improve SCI outcomes by directly or indirectly promoting an environment conductive to revascularization of the injured spinal cord, thus promoting neuronal regeneration.

According to Bartholdi and Schwab (Bartholdi and Schwab, 1995), the primary traumatic mechanical injury to the spinal cord causes the death of a number of neurons. However, the neurons continued to die at 4 h after SCI. The local inflammatory response in the injured spinal cord is believed to contribute significantly to the evolution of secondary damage. There is evidence that early inflammatory events promote tissue damage in the acutely injured spinal cord (Popovich and Jones, 2003). The inflammatory response, characterized by the infiltration of neutrophils and the activation of microglia, develops within hours after SCI (McTigue et al., 2000). This is followed by the expression of proinflammatory cytokines, including TNF-α and IL-1β, at the site of injury (Streit et al., 1998). In the present study, we further observed that HBO₂ treatment reduces these early inflammatory events in SCI rats, and we proposed that the attenuation of the early inflammatory response reduced secondary damage. This observation is in agreement with the results of other studies, that have clearly demonstrated that etanercept, a TNF-α inhibitor, significantly reduces SCI-induced spinal cord tissue damage, and improves hindlimb locomotor function (Genovese et al., 2006).

Finally, it should be stressed that in the current study, the fact that HBO₂ was administered immediately after SCI makes translation into the clinical setting unlikely. Initiating HBO₂ therapy soon after spinal cord injury deserves further study.

Footnotes

Acknowledgments

We would like to thank the Department of Medical Research of the Chi Mei Medical Center (Tainan, Taiwan) for providing laboratory support. This research was funded in part by the National Science Council of the Republic of China (grants NSC96-2314-B-384-006-MY3 and NSC96-2314-B-384-003-MY3) and DOH99-TD-B-III-003 Center of Excellence for Clinical Trial and Research in Neuroscience.

Author Disclosure Statement

No competing financial interests exist.

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