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Effects of progesterone on experimental spinal cord injury
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Effects of progesterone on experimental spinal cord injury Dominic B. Fee a,b,⁎, Karin R. Swartz b,c , Kelly M. Joy d , Kelly N. Roberts d , Nicole N. Scheff d , Stephen W. Scheff b,d a
Department of Neurology, University of Kentucky Chandler Medical Center, Lexington, KY 40536, USA Spinal Cord and Brain Injury Research Center, University of Kentucky Chandler Medical Center, Lexington, KY 40536, USA c Division of Neurosurgery, University of Kentucky Chandler Medical Center, Lexington, KY 40536, USA d Sanders–Brown Center on Aging; University of Kentucky Chandler Medical Center, Lexington, KY 40536, USA b
A R T I C LE I N FO
AB S T R A C T
Article history:
Progesterone has been proposed to be protective to the central nervous system following
Accepted 12 December 2006
injury. This study assessed progesterone supplementation in the setting of contusional
Available online 3 January 2007
spinal cord injury in male and female rats. Short-term (5 days of either 4 or 8 mg/kg progesterone) and long-term (14 days of either 8 or 16 mg/kg progesterone) therapy failed to
Keywords:
show any significant alteration in locomotor functioning and injury morphometrics after
Progesterone
21 days. This study does not support progesterone as a potential therapeutic agent in spinal
Spinal cord injury
cord injury.
Contusion
© 2006 Elsevier B.V. All rights reserved.
Neurotrauma Gender BBB
1.
Introduction
There is growing literature suggesting that progesterone (PG) may be a neuroprotective agent following a variety of central nervous system injuries, including trauma and stroke (Betz and Coester, 1990; Roof et al., 1994; Jiang et al., 1996; Roof et al., 1996; Goss et al., 2003; Djebaili et al., 2004). PG receptors are widely distributed throughout the central nervous system (CNS) including the spinal cord (SC) (MacLusky and McEwen, 1978; Labombarda et al., 2003), and de novo synthesis within the CNS can occur independent of gonadal secretion (Robel and Baulieu, 1995; Schumacher et al., 2004). Within the SC, PG has been shown to improve lower motor neuron survival in neurodegenerative and peripheral axonotomesis models (Yu,
1989; Gonzalez Deniselle et al., 2002). Furthermore, following percussion-induced spinal cord injury (SCI), PG production at and around the site of injury can be increased by pregnenolone therapy (di Michele et al., 2000). Pregnenolone when added to other therapeutic agents, has been shown to significantly enhance recovery following SCI; however, pregnenolone alone had no effect on SCI (Guth et al., 1994). Also, PG has been reported to modulate spinal cord-mediated pain responses (Gintzler and Liu, 2001). These results suggest that PG may have an intrinsic SC neuromodulatory/neurotrophic function. There is only one paper directly assessing PG supplementation on SC damage and hind limb recovery following traumatic SCI. Thomas et al. assessed short duration (5 days), post-injury
⁎ Corresponding author. Department of Neurology, KY Clinic, Rm L-445, Lexington, KY 40536-0284, USA. Fax: +1 859 323 5943. E-mail address: [email protected] (D.B. Fee). Abbreviations: PG, progesterone; SC, spinal cord; SCI, spinal cord injury; BBB, Basso, Beattie, Bresnahan Locomotor Rating Scale; WM, white matter; GM, grey matter; CNS, central nervous system 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.12.024
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PG therapy in rats following a moderate SCI; they reported that the PG therapy improved Basso, Beattie, Bresnehan Locomotor Rating Scale (BBB) scores, as well as increased sparing of white matter (WM) in the injured SC (Thomas et al., 1999). There are many articles attempting to determine the mechanism of this proposed benefit using a SC transection model assessing tissue caudal to the injury (Labombarda et al., 2000, 2002, 2003; Gonzalez et al., 2004, 2005). The Thomas et al. study was limited to a single PG dosage for 5 days of supplementation and to male rats (Thomas et al., 1999), leaving open the question of whether the PG-related benefits would be present in females, and if outcomes could be further improved with increased dose and/or protracted therapy. We designed a study to try to determine the optimal progesterone supplementation strategy for male and female rats; the goal was not to exactly replicate the Thomas et al. study. We did mirror the 4 mg/kg for 5-day supplementation in male rats as done in Thomas et al., as well as added an 8 mg/kg for 5-day regimen and assessed female rats. Furthermore, we assessed 8 mg/kg and 16 mg/kg progesterone supplementation for 14 days in both male and female rats. We included the second regimen because other studies have reported that high concentration PG supplementation in the injured peripheral nervous system promotes long-term myelin formation (Koenig et al., 1995) and PG has also been shown to enhance CNS remyelination (Ghoumari et al., 2003; Ibanez et al., 2003); both events are needed to help bridge injured SC.
2.
Results
2.1.
Locomotor recovery (BBB)
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The effect of PG on locomotor function was evaluated to determine if any benefit was seen among the same sex animal. BBB testing was performed at post-injury days 2, 7, 14, and 21. All animals demonstrated significant hind limb locomotor impairment after SCI, followed by significant improvement over the subsequent 21 days, the majority by day 7 (Figs. 1A–D).
2.1.1.
5-Day supplementation arm
In the 5-day supplementation arm, a two-way ANOVA (TREATMENT by GENDER) at day 2 post-SCI failed to detect a significant PG [F(2,32) = 0.035; p > 0.1] or GENDER [F(1,32) = 2.188; p > 0.1] effect, indicating equivalent locomotor impairment between groups. Additional analysis at days 7, 14, and 21 post-SCI also failed to detect any PG or gender effects. At day 21, the two-way ANOVA was PG [F(2,32) = 0.202; p > 0.1] and GENDER [F(1,32) = 0.759; p > 0.1] indicating that all groups attained similar levels of recovery; the ANOVA analysis for days 7 and 14 is not shown.
2.1.2.
14-Day supplementation arm
In the 14-day supplementation arm, a two-way ANOVA (TREATMENT by GENDER) at day 2 post-SCI did not detect any significant PG [F(2,31) = 0.176; p > 0.1] or GENDER [F(1,31) =
Fig. 1 – Mean locomotor behavioral scores (BBB) for both genders in both supplementation arms. There are no significant differences between the supplemented and control rats. The bars below the data points indicate a standard deviation. Each group had 6–7 rats.
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1.009; p > 0.1] effect on locomotor impairment in the short term. Additional analysis at days 7, 14, and 21 post-SCI also failed to detect any PG or gender effects. At day 21, the twoway ANOVA was PG [F(2,31) = 0.691; p > 0.1] and GENDER [F(1,31) = 0.499; p > 0.1], again indicating that all groups attained a similar level of recovery in the long term; the ANOVA analysis for days 7 and 14 is not shown.
2.2.
Morphologic assessment
2.2.1.
5-Day supplementation arm
2.2.1.1. Injury length. The rostral–caudal extent of the injury was determined for each subject. A two-way ANOVA (TREATMENT by GENDER) failed to reveal a significant TREATMENT (PG) effect [F(2,32) = 1.797; p > 0.1] nor a significant GENDER effect [F(1,32) = 2.112; p > 0.1]. Post-injury progesterone therapy for 5 days did not reduce the length of the injury (Table 1). 2.2.1.2. Spared tissue. The percent of total spared tissue was assessed for each subject and group means were assessed with a two-way ANOVA (TREATMENT by GENDER). There was no significant TREATMENT effect [F(2,32) = 1.762; p > 0.1] for total tissue sparing, nor for GENDER effect [F(1,32) = 1.478; p > 0.1]. WM and GM volumes were also assessed. There was no treatment or gender effect seen with percent total spared WM, PG [F(2,32) = 0.856; p > 0.1] and GENDER [F(2,32) = 0.027; p > 0.1]. However, percent total spared GM did statistically worse with an increasing concentration of progesterone, with females trending worse than males, PG [F(2,32) = 4.230; p = 0.023] and GENDER [F(2,32) = 3.697; p = 0.063]. Tissue sparing at the injury epicenter also failed to show a significant TREATMENT effect [F(2,32) = 1.622; p > 0.1] or GENDER effect [F(1,32) = 1.549; p > 0.1]. 2.2.2.
(TREATMENT by GENDER) failed to reveal a significant PG effect [F(2,31) = 0.496; p > 0.1] or a significant GENDER effect [F(1,31) = 0.255; p > 0.1]. Post-injury progesterone therapy for 14 days did not reduce the length of the injury (Table 1).
2.2.2.2. Spared tissue. The percent of total spared tissue was assessed for each subject and group mean scores were assessed with a two-way ANOVA (TREATMENT by GENDER). There was no significant TREATMENT effect [F(2,31) = 0.156; p > 0.1] or GENDER effect [F(1,31) = 2.495; p > 0.1] for total tissue sparing. WM and GM volumes were also assessed. There was no treatment or gender effect seen with percent total spared WM, PG [F(2,31) = 0.207; p > 0.1] and GENDER [F(2,31) = 2.467; p > 0.1], and GM, PG [F(2,31) = 0.192; p > 0.1] and GENDER [F(2,31) = 2.175; p > 0.1]. Tissue sparing at the injury epicenter also failed to show a significant GENDER effect [F(1,31) = 0.007; p > 0.1]; however, there was a significant TREATMENT (PG) effect [F(2,31) = 4.097; p > 0.026] with greater sparing as progesterone concentration increased (Table 1). 2.2.3.
3.
14-Day supplementation arm
2.2.2.1. Injury length. The rostral–caudal extent of the injury was determined for each subject. A two-way ANOVA
Short-term vs. long-term supplementation
Although our initial assessment was to compare dosages within set days of supplementation, we wanted to also compare the results between the 5-day, 4 and 8 mg/kg and 14-day, 8 and 16 mg/kg supplementation groups. For both males and females, 14-day supplementation had shorter injury lengths as compared to 5-day supplementation, male [F(2,31) = 7.022; p = 0.013] and female [F(2,32) = 8.416; p = 0.007]. However, there was no difference in percent total spared tissue, male [F(2,31) = 1.129; p > 0.1] or female [F(2,32) = 1.428; p > 0.1]. Nor was there a difference in percent epicenter sparing for females [F(2,32) = 1.194; p > 0.1]; however, for males, the 14-day supplementation had worse percent spared epicenter, [F(2,31) = 4.991; p = 0.033].
Discussion
There are many studies suggesting that progesterone (PG) is neuroprotective/neuromodulatory to the CNS. In brain injury
Table 1 – Morphometric measurements Male 5-Day supplementation
Female
Control
4 mg/kg
8 mg/kg
Control
4 mg/kg
8 mg/kg
Injury length Epicenter % spared tissue % Total spared tissue % Total spared WM % Total spared GM
8167 ± 3488 46.20 ± 21.08 87.36 ± 6.81 90.68 ± 5.84 80.23 ± 10.69
11,000 ± 6000 55.37 ± 22.55 86.96 ± 8.37 92.26 ± 7.61 74.06 ± 16.61
10,667 ± 3933 56.62 ± 12.23 86.43 ± 7.36 91.12 ± 5.87 72.00 ± 16.18
9571 ± 4614 37.55 ± 20.32 89.8 ± 5.32 95.37 ± 1.77 80.31 ± 13.30
13,286 ± 6726 49.14 ± 13.44 83.00 ± 9.75 90.87 ± 10.88 63.88 ± 17.77 ⁎
14,800 ± 7259 49.99 ± 9.30 78.06 ± 11.57 86.57 ± 11.68 54.26 ± 10.97 ⁎
14-Day supplementation
Control
8 mg/kg
16 mg/kg
Control
8 mg/kg
16 mg/kg
Injury length Epicenter % spared tissue % Total spared tissue % Total spared WM % Total spared GM
6667 ± 2658 32.94 ± 15.93 90.27 ± 5.71 92.63 ± 4.99 82.87 ± 7.56
7333 ± 1366 41.02 ± 18.03 88.02 ± 4.29 92.07 ± 5.96 80.47 ± 2.98
6333 ± 1366 45.78 ± 9.88 89.00 ± 2.40 93.85 ± 3.53 83.24 ± 3.42
8571 ± 5711 37.66 ± 12.16 86.37 ± 5.51 89.65 ± 5.16 79.00 ± 8.81
5183 ± 2281 29.56 ± 16.95 87.74 ± 3.81 90.84 ± 3.39 81.93 ± 6.07
8367 ± 4948 53.71 ± 11.76 ⁎ 85.43 ± 6.72 90.77 ± 4.50 76.06 ± 7.80
Mean ± standard deviation. ∗ p < 0.05 vs. control.
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models, PG has been shown to be beneficial in stroke and trauma models (Betz and Coester, 1990; Roof et al., 1994; Jiang et al., 1996; Roof et al., 1996; Goss et al., 2003; Djebaili et al., 2004). Only one study, prior to this one, has directly assessed progesterone therapy in a spinal cord injury model (Thomas et al., 1999). Other studies have assessed mechanisms of the proposed benefit of progesterone therapy in SCI (Labombarda et al., 2000, 2002, 2003; Gonzalez et al., 2004, 2005). The present data failed to replicate the beneficial effects of progesterone in SCI seen by Thomas et al. (1999). We assessed the same dosage of PG and took our investigation further with a higher dosage for the same duration of treatment, 5 days; however, our SCI was less severe. A 150 kdyn impaction by an IH impacting device results in a SCI similar to one made by a 10 g bar at a height of 12.5 mm on an NYU device, and in general, the NYU device also resulted in greater WM loss (Rabchevsky et al., 2003; Scheff et al., 2003). Thomas et al. used an NYU device with a 10 g bar at 25 mm and, therefore, had a more severe injury as compared to the injury created in this study. In addition to differing doses, we also assessed female as well as male animals. There were no consistent treatment or gender differences, seen in locomotor (BBB) recovery nor in SC morphology, in our study. Thomas et al. assessed their rats at day 42 post-injury, rather than at day 21 post-injury in this study (Thomas et al., 1999); however, it is doubtful that this difference in end date would result in different conclusions. With regard to behavioral (BBB) assessment, one of their control groups, injury but no vehicle (termed “control”), follows a distinctly separate recovery as compared to their progesterone supplementation group beginning at day 3 post-injury and maintained this throughout the remainder of the study; the other control group, injury with vehicle (termed “DMSO”), distinctly separates by day 21 post-injury (Thomas et al., 1999). Although no statistical analysis was reported on BBB scores at earlier time points and the error bars provided are not defined, the data do indicate that there was a difference between the only progesterone supplementation group and the two injured control groups at day 21 post-injury. Additionally, the BBB scores for the progesterone supplementation group in Thomas et al. had continuous improvement at every time point assessed (Thomas et al., 1999); however, the male 5day, 4 mg/kg progesterone supplementation group in our study, which is identical to the sole experiment group in Thomas et al., plateaued by day 14 post-SCI and mirrored the control group. This plateau of BBB by day 14 post-SCI is consistent with longitudinal studies of BBB following SCI (Basso et al., 1996). With regard to the histologic data, Thomas et al. felt that progesterone was neuroprotective and exerted its effects on the injury response which occurred within days of the SCI. Therefore, if this is true, then the improved percent intact white matter at the epicenter seen in the progesterone supplementation arm of Thomas et al. would have been present at any time point following the acute injury phase, which would include day 21 post-injury. Our histological assessment included white matter at the epicenter as well as other measures assessing two-dimensional and three-dimensional aspects of the injury; no benefit was seen. Furthermore, there have been studies attempting
149
to determine the mechanism of the proposed neuroprotective effects of progesterone following SCI; these have assessed the injuries at 72 or 75 h post-injury(Labombarda et al., 2000, 2002, 2003; Gonzalez et al., 2004, 2005). Again, the studies proposing a progesterone benefit in SCI indicate that this occurs in the acute injury phase; therefore, neuroprotective effects resulting in preserved tissue should be present by day 21 post-SCI, if not sooner, rather than developing late in the chronic injury phase. Finally, our study also assessed a higher dosage (8 mg/kg) 5-day progesterone supplementation with no difference from control seen, i.e., no evidence for dose responsiveness was appreciated. In a second experimental design, we extended the treatment for an additional 9 days (14 days total) and used two higher dosages of PG. Again, these results failed to show a consistent PG or gender effect. The only potential benefit was in the 14-day supplementation group. The 16 mg/kg group had significantly better epicenter sparing; however, this did not translate into improved total spared tissue nor behavioral outcomes. This discrepancy may reflect slight differences in the shape of the injury, but this did not change the volume of the injury, which is the most significant morphologic variable, nor did it change locomotor recovery. Contrary to the notion that PG is neuroprotective, there are data from our study suggesting that progesterone maybe deleterious; in the 5-day supplementation arm, progesterone supplementation was associated with a worsening of percent spared GM. Again, neither of these two findings were supported by other measured parameters in this study and are probably an epiphenomenon. Similarly, comparing the 5-day and 14-day supplementation arms, although certain morphometric measurements showed some differences, total percent spared tissue did not. Progesterone has been proposed to be neuroprotective to motor neurons, elements of GM (Yu, 1989; Gonzalez Deniselle et al., 2002). Our study did not show any effect on GM sparing in the 14-day supplementation arm. In the 5-day supplementation arm, higher dosages of progesterone led to less preserved GM, especially in females. This difference maybe related to experimental design: the cited studies used neurodegenerative and peripheral nerve axonotomesis models, whereas we used a contusional injury and measured regions rather than cells. With our data, the only gender or dose responsiveness to progesterone supplementation was with worsened percent spared GM as progesterone increased, seen in the 5-day arm, and with improved epicenter sparing, seen with higher dosages of progesterone in the 14-day arm. However, these findings were not supported by other morphometric measurements nor consistent between the two experimental designs. Although we set out to determine optimal progesterone supplementation in SCI, our data, overall, suggest that contusional spinal cord injury is indifferent to progesterone. In comparing this work to Thomas et al., the only two significant differences between our male, 5-day supplementation arm and the sole experimental group in Thomas et al. were that the animals in Thomas et al. were sacrificed at 6 weeks post-SCI rather than 3 weeks and had a more severe injury (Thomas et al., 1999). However, we doubt that these differences would explain the disparate findings.
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4.
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Experimental procedures
All protocols were in accordance with national animal care guidelines and were carried out after approval from the University of Kentucky Animal Care and Use Committee.
4.1.
Spinal cord injury
Six- to eight-week-old adult male (285–300 g) and female (192– 268 g) Sprague-Dawley rats were used for these studies. All animals undergoing surgery were anesthetized with ketamine (80 mg/kg, Fort Dodge Animal Health; Fort Dodge, IS) and xylazine (10 mg/kg, The Butler Company; Columbus, OH) before undergoing a laminectomy at the T10 vertebral level and receiving a moderate (150 kdyn) spinal cord contusion using the well-characterized IH impacting device (PSI, Fairfax, VA) (Rabchevsky et al., 2003; Scheff et al., 2003). A 2.5 mm impactor tip was set 8 mm above the spinal cord. After impact, the wounds were irrigated with saline, and the muscle and fascia were reapproximated with absorbable sutures. The skin was closed with surgical staples. The animals had their temperature maintained at a constant level with heating pads during the surgery and post-operative period.
4.2.
Post-injury care
Each rat received 10 ml sterile saline injected subcutaneously and 33.3 mg/kg cefazolin (SoloPak Laboratories; Elk Grove, IL) intramuscularly, immediately post-operation, as well as on post-injury days 1 and 2; additional cefazolin was given for any evidence of hematuria/urinary tract infection. Bladders were manually expressed twice daily using the method of Crede until bladder function returned. Animals had access to food and water ad libitum. The rats were housed under the auspices of the University of Kentucky Animal Care Facility.
4.3.
Experimental design
4.3.1.
5-Day supplementation arm
Male (n = 19) and female (n = 19) rats were assigned to one of three different treatment groups: vehicle (dimethylsulfoxide, DMSO (Sigma; St. Louis, MO)), 4 mg/kg PG, and 8 mg/kg
progesterone (PG (P8783, Sigma; St. Louis, MO)). Following the contusional SCI, animals received PG therapy at 30 min, 6 h and once daily for 5 days. This paradigm recreates the one used by Thomas et al. (1999) and adds an 8 mg/kg group.
4.3.2.
14-Day supplementation arm
Male (n = 18) and female (n = 19) rats were assigned to one of three different treatment groups: vehicle (hydroxypropyl-βcyclodextrin (Sigma; St. Louis, MO)), 8 mg/kg PG, and 16 mg/kg PG. Following SCI, animals received PG therapy at 30 min, 6 h, and once daily for 14 days post-injury.
4.4.
Behavioral analyses
To examine possible effects of progesterone on locomotor recovery, animals were tested and scored using the Basso, Beattie, Bresnahan Locomotor Rating Scale (BBB) (Basso et al., 1996). Animal activity was scored by at least two trained individuals who were blinded to the treatments of each animal. Scoring discrepancies were discussed by the evaluators at the time of testing; averages were taken. BBB scores were generated for each hind limb, with the average of the two limbs (rounded down) tabulated at post-injury days 2, 7, 14, and 21; the rats were assessed pre-injury to assure no baseline deficits. Mean scores were calculated for each treatment group with the differences investigated using analysis of variance (ANOVA) via StatView 4.5.3 statistical package (Abacus Concepts Inc: Berkeley, CA) (Scheff et al., 2002). Significance was set at p < 0.05 for all experiments.
4.5.
Spinal cord harvesting and processing
At day 21 post-injury, animals were overdosed with sodium pentobarbital (Abbott Laboratories; North Chicago, IL) and transcardially perfused with 0.1 M phosphate-buffered saline (PBS, pH 7.4) and 4% paraformaldehyde (Sigma; St. Louis, MO) in PBS. SC was dissected away from surround tissue and bone from T6-L1, as a 3 cm segment. The SC was then additionally fixed in 4% paraformaldehyde for 24 h, washed in PBS for 24 h, then cryoprotected in 20% sucrose/PBS at 4 °C prior to embedding in gum tragacanth (Sigma; St. Louis, MO) and snap frozen in acetone cooled on dry ice. The embedded spinal cords were stored at −80 °C until sectioning. The embedded SC
Fig. 2 – Representative images of the spinal cord damage following a 150 kdyn contusional injury. Image B is the injury epicenter; the other images are the next section, 1 mm distant, cranial (A) and caudal (C) to the epicenter. The bar indicates 1 mm.
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were serially cross-sectioned at 20 μm in a Jung Cryostat set at −25 °C. Every fifth section of 20 μm thickness was placed, sequentially, on slides numbered 1–10; thusly each slide contained sections 1 mm apart. Ten sets of slides, each with about 30 sections, 1 mm apart, were available for morphometric analysis. The slides were stored at − 20 °C until use.
4.6.
Staining and morphometric analysis
The slides were stained with a modified eriochrome cyanine (EC) staining protocol which allowed for differentiation of white matter and grey matter (Rabchevsky et al., 2001). Stereologic image analysis was performed on the EC-stained sections using NIH Image v1.62 (NIH; Betheseda, MD). To minimize differences between animals, the ten sections (10 mm) cranial to the epicenter, and the ten sections caudal to the epicenter were used for analysis. At least two sets of sections were available for review per spinal cord. Length of injury was determined by taking the number of sections with identifiable injury present and multiplying it by 1000 μm (distance between sections). Spared tissue was determined by the presence of preserved architecture with the EC staining of myelin in white matter (WM) and cell body staining in grey matter (GM). The epicenter was defined as the section with the least percentage of spared tissue (Fig. 2). Nearly all the persevered tissue at the epicenter is WM; an inconsequential amount is GM. Therefore, percent spared tissue at the epicenter reflects preserved WM which correlates to the histologic measurements in Thomas et al. The volume assessments were calculated assuming linear gradation and 1 mm distance between section and using the Cavlieri method (Rabchevsky et al., 2001). Total WM volume was obtained by subtracting total GM volume and total injury volume from total volume (Rabchevsky et al., 2000). Predicted total volume, predicted GM volume, and predicted WM volume were determined by averaging the total area, GM area, and WM area in five uninjured sections rostral and caudal to the injury; these results were used to extrapolate the pre-injury areas in the injured SC segments (Rabchevsky et al., 2001). Because females have smaller SC than males, percent spared tissue was calculated to allow for gender comparisons. These were calculated by dividing actual volumes by predicted volumes.
Acknowledgment The research was funded in part by the Kentucky Spinal Cord and Head Injury Trust #05A.
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Rabchevsky, A.G., Fugaccia, I., Sullivan, P.G., Scheff, S.W., 2001. Cyclosporin A treatment following spinal cord injury to the rat: behavioral effects and stereological assessment of tissue sparing. J. Neurotrauma 18, 513–522. Rabchevsky, A.G., Sullivan, P.G., Fugaccia, I., Scheff, S.W., 2003. Creatine diet supplement for spinal cord injury: influences on functional recovery and tissue sparing in rats. J. Neurotrauma 20, 659–669. Robel, P., Baulieu, E.E., 1995. Neurosteroids: biosynthesis and function. Crit. Rev. Neurobiol. 9, 383–394. Roof, R.L., Duvdevani, R., Braswell, L., Stein, D.G., 1994. Progesterone facilitates cognitive recovery and reduces secondary neuronal loss caused by cortical contusion injury in male rats. Exp. Neurol. 129, 64–69. Roof, R.L., Duvdevani, R., Heyburn, J.W., Stein, D.G., 1996. Progesterone rapidly decreases brain edema: treatment delayed up to 24 hours is still effective. Exp. Neurol. 138, 246–251. Scheff, S.W., Saucier, D.A., Cain, M.E., 2002. A statistical method for
analyzing rating scale data: the BBB locomotor score. J. Neurotrauma 19, 1251–1260. Scheff, S.W., Rabchevsky, A.G., Fugaccia, I., Main, J.A., Lumpp Jr., J.E., 2003. Experimental modeling of spinal cord injury: characterization of a force-defined injury device. J. Neurotrauma 20, 179–193. Schumacher, M., Guennoun, R., Robert, F., Carelli, C., Gago, N., Ghoumari, A., Gonzalez Deniselle, M.C., Gonzalez, S.L., Ibanez, C., Labombarda, F., Coirini, H., Baulieu, E.E., De Nicola, A.F., 2004. Local synthesis and dual actions of progesterone in the nervous system: neuroprotection and myelination. Growth Horm. IGF Res. 14 (Suppl. A), S18–S33. Thomas, A.J., Nockels, R.P., Pan, H.Q., Shaffrey, C.I., Chopp, M., 1999. Progesterone is neuroprotective after acute experimental spinal cord trauma in rats. Spine 24, 2134–2138. Yu, W.H., 1989. Survival of motoneurons following axotomy is enhanced by lactation or by progesterone treatment. Brain Res. 491, 379–382.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Effects of progesterone on experimental spinal cord injury Dominic B. Fee a,b,⁎, Karin R. Swartz b,c , Kelly M. Joy d , Kelly N. Roberts d , Nicole N. Scheff d , Stephen W. Scheff b,d a
Department of Neurology, University of Kentucky Chandler Medical Center, Lexington, KY 40536, USA Spinal Cord and Brain Injury Research Center, University of Kentucky Chandler Medical Center, Lexington, KY 40536, USA c Division of Neurosurgery, University of Kentucky Chandler Medical Center, Lexington, KY 40536, USA d Sanders–Brown Center on Aging; University of Kentucky Chandler Medical Center, Lexington, KY 40536, USA b
A R T I C LE I N FO
AB S T R A C T
Article history:
Progesterone has been proposed to be protective to the central nervous system following
Accepted 12 December 2006
injury. This study assessed progesterone supplementation in the setting of contusional
Available online 3 January 2007
spinal cord injury in male and female rats. Short-term (5 days of either 4 or 8 mg/kg progesterone) and long-term (14 days of either 8 or 16 mg/kg progesterone) therapy failed to
Keywords:
show any significant alteration in locomotor functioning and injury morphometrics after
Progesterone
21 days. This study does not support progesterone as a potential therapeutic agent in spinal
Spinal cord injury
cord injury.
Contusion
© 2006 Elsevier B.V. All rights reserved.
Neurotrauma Gender BBB
1.
Introduction
There is growing literature suggesting that progesterone (PG) may be a neuroprotective agent following a variety of central nervous system injuries, including trauma and stroke (Betz and Coester, 1990; Roof et al., 1994; Jiang et al., 1996; Roof et al., 1996; Goss et al., 2003; Djebaili et al., 2004). PG receptors are widely distributed throughout the central nervous system (CNS) including the spinal cord (SC) (MacLusky and McEwen, 1978; Labombarda et al., 2003), and de novo synthesis within the CNS can occur independent of gonadal secretion (Robel and Baulieu, 1995; Schumacher et al., 2004). Within the SC, PG has been shown to improve lower motor neuron survival in neurodegenerative and peripheral axonotomesis models (Yu,
1989; Gonzalez Deniselle et al., 2002). Furthermore, following percussion-induced spinal cord injury (SCI), PG production at and around the site of injury can be increased by pregnenolone therapy (di Michele et al., 2000). Pregnenolone when added to other therapeutic agents, has been shown to significantly enhance recovery following SCI; however, pregnenolone alone had no effect on SCI (Guth et al., 1994). Also, PG has been reported to modulate spinal cord-mediated pain responses (Gintzler and Liu, 2001). These results suggest that PG may have an intrinsic SC neuromodulatory/neurotrophic function. There is only one paper directly assessing PG supplementation on SC damage and hind limb recovery following traumatic SCI. Thomas et al. assessed short duration (5 days), post-injury
⁎ Corresponding author. Department of Neurology, KY Clinic, Rm L-445, Lexington, KY 40536-0284, USA. Fax: +1 859 323 5943. E-mail address: [email protected] (D.B. Fee). Abbreviations: PG, progesterone; SC, spinal cord; SCI, spinal cord injury; BBB, Basso, Beattie, Bresnahan Locomotor Rating Scale; WM, white matter; GM, grey matter; CNS, central nervous system 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.12.024
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PG therapy in rats following a moderate SCI; they reported that the PG therapy improved Basso, Beattie, Bresnehan Locomotor Rating Scale (BBB) scores, as well as increased sparing of white matter (WM) in the injured SC (Thomas et al., 1999). There are many articles attempting to determine the mechanism of this proposed benefit using a SC transection model assessing tissue caudal to the injury (Labombarda et al., 2000, 2002, 2003; Gonzalez et al., 2004, 2005). The Thomas et al. study was limited to a single PG dosage for 5 days of supplementation and to male rats (Thomas et al., 1999), leaving open the question of whether the PG-related benefits would be present in females, and if outcomes could be further improved with increased dose and/or protracted therapy. We designed a study to try to determine the optimal progesterone supplementation strategy for male and female rats; the goal was not to exactly replicate the Thomas et al. study. We did mirror the 4 mg/kg for 5-day supplementation in male rats as done in Thomas et al., as well as added an 8 mg/kg for 5-day regimen and assessed female rats. Furthermore, we assessed 8 mg/kg and 16 mg/kg progesterone supplementation for 14 days in both male and female rats. We included the second regimen because other studies have reported that high concentration PG supplementation in the injured peripheral nervous system promotes long-term myelin formation (Koenig et al., 1995) and PG has also been shown to enhance CNS remyelination (Ghoumari et al., 2003; Ibanez et al., 2003); both events are needed to help bridge injured SC.
2.
Results
2.1.
Locomotor recovery (BBB)
147
The effect of PG on locomotor function was evaluated to determine if any benefit was seen among the same sex animal. BBB testing was performed at post-injury days 2, 7, 14, and 21. All animals demonstrated significant hind limb locomotor impairment after SCI, followed by significant improvement over the subsequent 21 days, the majority by day 7 (Figs. 1A–D).
2.1.1.
5-Day supplementation arm
In the 5-day supplementation arm, a two-way ANOVA (TREATMENT by GENDER) at day 2 post-SCI failed to detect a significant PG [F(2,32) = 0.035; p > 0.1] or GENDER [F(1,32) = 2.188; p > 0.1] effect, indicating equivalent locomotor impairment between groups. Additional analysis at days 7, 14, and 21 post-SCI also failed to detect any PG or gender effects. At day 21, the two-way ANOVA was PG [F(2,32) = 0.202; p > 0.1] and GENDER [F(1,32) = 0.759; p > 0.1] indicating that all groups attained similar levels of recovery; the ANOVA analysis for days 7 and 14 is not shown.
2.1.2.
14-Day supplementation arm
In the 14-day supplementation arm, a two-way ANOVA (TREATMENT by GENDER) at day 2 post-SCI did not detect any significant PG [F(2,31) = 0.176; p > 0.1] or GENDER [F(1,31) =
Fig. 1 – Mean locomotor behavioral scores (BBB) for both genders in both supplementation arms. There are no significant differences between the supplemented and control rats. The bars below the data points indicate a standard deviation. Each group had 6–7 rats.
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1.009; p > 0.1] effect on locomotor impairment in the short term. Additional analysis at days 7, 14, and 21 post-SCI also failed to detect any PG or gender effects. At day 21, the twoway ANOVA was PG [F(2,31) = 0.691; p > 0.1] and GENDER [F(1,31) = 0.499; p > 0.1], again indicating that all groups attained a similar level of recovery in the long term; the ANOVA analysis for days 7 and 14 is not shown.
2.2.
Morphologic assessment
2.2.1.
5-Day supplementation arm
2.2.1.1. Injury length. The rostral–caudal extent of the injury was determined for each subject. A two-way ANOVA (TREATMENT by GENDER) failed to reveal a significant TREATMENT (PG) effect [F(2,32) = 1.797; p > 0.1] nor a significant GENDER effect [F(1,32) = 2.112; p > 0.1]. Post-injury progesterone therapy for 5 days did not reduce the length of the injury (Table 1). 2.2.1.2. Spared tissue. The percent of total spared tissue was assessed for each subject and group means were assessed with a two-way ANOVA (TREATMENT by GENDER). There was no significant TREATMENT effect [F(2,32) = 1.762; p > 0.1] for total tissue sparing, nor for GENDER effect [F(1,32) = 1.478; p > 0.1]. WM and GM volumes were also assessed. There was no treatment or gender effect seen with percent total spared WM, PG [F(2,32) = 0.856; p > 0.1] and GENDER [F(2,32) = 0.027; p > 0.1]. However, percent total spared GM did statistically worse with an increasing concentration of progesterone, with females trending worse than males, PG [F(2,32) = 4.230; p = 0.023] and GENDER [F(2,32) = 3.697; p = 0.063]. Tissue sparing at the injury epicenter also failed to show a significant TREATMENT effect [F(2,32) = 1.622; p > 0.1] or GENDER effect [F(1,32) = 1.549; p > 0.1]. 2.2.2.
(TREATMENT by GENDER) failed to reveal a significant PG effect [F(2,31) = 0.496; p > 0.1] or a significant GENDER effect [F(1,31) = 0.255; p > 0.1]. Post-injury progesterone therapy for 14 days did not reduce the length of the injury (Table 1).
2.2.2.2. Spared tissue. The percent of total spared tissue was assessed for each subject and group mean scores were assessed with a two-way ANOVA (TREATMENT by GENDER). There was no significant TREATMENT effect [F(2,31) = 0.156; p > 0.1] or GENDER effect [F(1,31) = 2.495; p > 0.1] for total tissue sparing. WM and GM volumes were also assessed. There was no treatment or gender effect seen with percent total spared WM, PG [F(2,31) = 0.207; p > 0.1] and GENDER [F(2,31) = 2.467; p > 0.1], and GM, PG [F(2,31) = 0.192; p > 0.1] and GENDER [F(2,31) = 2.175; p > 0.1]. Tissue sparing at the injury epicenter also failed to show a significant GENDER effect [F(1,31) = 0.007; p > 0.1]; however, there was a significant TREATMENT (PG) effect [F(2,31) = 4.097; p > 0.026] with greater sparing as progesterone concentration increased (Table 1). 2.2.3.
3.
14-Day supplementation arm
2.2.2.1. Injury length. The rostral–caudal extent of the injury was determined for each subject. A two-way ANOVA
Short-term vs. long-term supplementation
Although our initial assessment was to compare dosages within set days of supplementation, we wanted to also compare the results between the 5-day, 4 and 8 mg/kg and 14-day, 8 and 16 mg/kg supplementation groups. For both males and females, 14-day supplementation had shorter injury lengths as compared to 5-day supplementation, male [F(2,31) = 7.022; p = 0.013] and female [F(2,32) = 8.416; p = 0.007]. However, there was no difference in percent total spared tissue, male [F(2,31) = 1.129; p > 0.1] or female [F(2,32) = 1.428; p > 0.1]. Nor was there a difference in percent epicenter sparing for females [F(2,32) = 1.194; p > 0.1]; however, for males, the 14-day supplementation had worse percent spared epicenter, [F(2,31) = 4.991; p = 0.033].
Discussion
There are many studies suggesting that progesterone (PG) is neuroprotective/neuromodulatory to the CNS. In brain injury
Table 1 – Morphometric measurements Male 5-Day supplementation
Female
Control
4 mg/kg
8 mg/kg
Control
4 mg/kg
8 mg/kg
Injury length Epicenter % spared tissue % Total spared tissue % Total spared WM % Total spared GM
8167 ± 3488 46.20 ± 21.08 87.36 ± 6.81 90.68 ± 5.84 80.23 ± 10.69
11,000 ± 6000 55.37 ± 22.55 86.96 ± 8.37 92.26 ± 7.61 74.06 ± 16.61
10,667 ± 3933 56.62 ± 12.23 86.43 ± 7.36 91.12 ± 5.87 72.00 ± 16.18
9571 ± 4614 37.55 ± 20.32 89.8 ± 5.32 95.37 ± 1.77 80.31 ± 13.30
13,286 ± 6726 49.14 ± 13.44 83.00 ± 9.75 90.87 ± 10.88 63.88 ± 17.77 ⁎
14,800 ± 7259 49.99 ± 9.30 78.06 ± 11.57 86.57 ± 11.68 54.26 ± 10.97 ⁎
14-Day supplementation
Control
8 mg/kg
16 mg/kg
Control
8 mg/kg
16 mg/kg
Injury length Epicenter % spared tissue % Total spared tissue % Total spared WM % Total spared GM
6667 ± 2658 32.94 ± 15.93 90.27 ± 5.71 92.63 ± 4.99 82.87 ± 7.56
7333 ± 1366 41.02 ± 18.03 88.02 ± 4.29 92.07 ± 5.96 80.47 ± 2.98
6333 ± 1366 45.78 ± 9.88 89.00 ± 2.40 93.85 ± 3.53 83.24 ± 3.42
8571 ± 5711 37.66 ± 12.16 86.37 ± 5.51 89.65 ± 5.16 79.00 ± 8.81
5183 ± 2281 29.56 ± 16.95 87.74 ± 3.81 90.84 ± 3.39 81.93 ± 6.07
8367 ± 4948 53.71 ± 11.76 ⁎ 85.43 ± 6.72 90.77 ± 4.50 76.06 ± 7.80
Mean ± standard deviation. ∗ p < 0.05 vs. control.
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models, PG has been shown to be beneficial in stroke and trauma models (Betz and Coester, 1990; Roof et al., 1994; Jiang et al., 1996; Roof et al., 1996; Goss et al., 2003; Djebaili et al., 2004). Only one study, prior to this one, has directly assessed progesterone therapy in a spinal cord injury model (Thomas et al., 1999). Other studies have assessed mechanisms of the proposed benefit of progesterone therapy in SCI (Labombarda et al., 2000, 2002, 2003; Gonzalez et al., 2004, 2005). The present data failed to replicate the beneficial effects of progesterone in SCI seen by Thomas et al. (1999). We assessed the same dosage of PG and took our investigation further with a higher dosage for the same duration of treatment, 5 days; however, our SCI was less severe. A 150 kdyn impaction by an IH impacting device results in a SCI similar to one made by a 10 g bar at a height of 12.5 mm on an NYU device, and in general, the NYU device also resulted in greater WM loss (Rabchevsky et al., 2003; Scheff et al., 2003). Thomas et al. used an NYU device with a 10 g bar at 25 mm and, therefore, had a more severe injury as compared to the injury created in this study. In addition to differing doses, we also assessed female as well as male animals. There were no consistent treatment or gender differences, seen in locomotor (BBB) recovery nor in SC morphology, in our study. Thomas et al. assessed their rats at day 42 post-injury, rather than at day 21 post-injury in this study (Thomas et al., 1999); however, it is doubtful that this difference in end date would result in different conclusions. With regard to behavioral (BBB) assessment, one of their control groups, injury but no vehicle (termed “control”), follows a distinctly separate recovery as compared to their progesterone supplementation group beginning at day 3 post-injury and maintained this throughout the remainder of the study; the other control group, injury with vehicle (termed “DMSO”), distinctly separates by day 21 post-injury (Thomas et al., 1999). Although no statistical analysis was reported on BBB scores at earlier time points and the error bars provided are not defined, the data do indicate that there was a difference between the only progesterone supplementation group and the two injured control groups at day 21 post-injury. Additionally, the BBB scores for the progesterone supplementation group in Thomas et al. had continuous improvement at every time point assessed (Thomas et al., 1999); however, the male 5day, 4 mg/kg progesterone supplementation group in our study, which is identical to the sole experiment group in Thomas et al., plateaued by day 14 post-SCI and mirrored the control group. This plateau of BBB by day 14 post-SCI is consistent with longitudinal studies of BBB following SCI (Basso et al., 1996). With regard to the histologic data, Thomas et al. felt that progesterone was neuroprotective and exerted its effects on the injury response which occurred within days of the SCI. Therefore, if this is true, then the improved percent intact white matter at the epicenter seen in the progesterone supplementation arm of Thomas et al. would have been present at any time point following the acute injury phase, which would include day 21 post-injury. Our histological assessment included white matter at the epicenter as well as other measures assessing two-dimensional and three-dimensional aspects of the injury; no benefit was seen. Furthermore, there have been studies attempting
149
to determine the mechanism of the proposed neuroprotective effects of progesterone following SCI; these have assessed the injuries at 72 or 75 h post-injury(Labombarda et al., 2000, 2002, 2003; Gonzalez et al., 2004, 2005). Again, the studies proposing a progesterone benefit in SCI indicate that this occurs in the acute injury phase; therefore, neuroprotective effects resulting in preserved tissue should be present by day 21 post-SCI, if not sooner, rather than developing late in the chronic injury phase. Finally, our study also assessed a higher dosage (8 mg/kg) 5-day progesterone supplementation with no difference from control seen, i.e., no evidence for dose responsiveness was appreciated. In a second experimental design, we extended the treatment for an additional 9 days (14 days total) and used two higher dosages of PG. Again, these results failed to show a consistent PG or gender effect. The only potential benefit was in the 14-day supplementation group. The 16 mg/kg group had significantly better epicenter sparing; however, this did not translate into improved total spared tissue nor behavioral outcomes. This discrepancy may reflect slight differences in the shape of the injury, but this did not change the volume of the injury, which is the most significant morphologic variable, nor did it change locomotor recovery. Contrary to the notion that PG is neuroprotective, there are data from our study suggesting that progesterone maybe deleterious; in the 5-day supplementation arm, progesterone supplementation was associated with a worsening of percent spared GM. Again, neither of these two findings were supported by other measured parameters in this study and are probably an epiphenomenon. Similarly, comparing the 5-day and 14-day supplementation arms, although certain morphometric measurements showed some differences, total percent spared tissue did not. Progesterone has been proposed to be neuroprotective to motor neurons, elements of GM (Yu, 1989; Gonzalez Deniselle et al., 2002). Our study did not show any effect on GM sparing in the 14-day supplementation arm. In the 5-day supplementation arm, higher dosages of progesterone led to less preserved GM, especially in females. This difference maybe related to experimental design: the cited studies used neurodegenerative and peripheral nerve axonotomesis models, whereas we used a contusional injury and measured regions rather than cells. With our data, the only gender or dose responsiveness to progesterone supplementation was with worsened percent spared GM as progesterone increased, seen in the 5-day arm, and with improved epicenter sparing, seen with higher dosages of progesterone in the 14-day arm. However, these findings were not supported by other morphometric measurements nor consistent between the two experimental designs. Although we set out to determine optimal progesterone supplementation in SCI, our data, overall, suggest that contusional spinal cord injury is indifferent to progesterone. In comparing this work to Thomas et al., the only two significant differences between our male, 5-day supplementation arm and the sole experimental group in Thomas et al. were that the animals in Thomas et al. were sacrificed at 6 weeks post-SCI rather than 3 weeks and had a more severe injury (Thomas et al., 1999). However, we doubt that these differences would explain the disparate findings.
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4.
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Experimental procedures
All protocols were in accordance with national animal care guidelines and were carried out after approval from the University of Kentucky Animal Care and Use Committee.
4.1.
Spinal cord injury
Six- to eight-week-old adult male (285–300 g) and female (192– 268 g) Sprague-Dawley rats were used for these studies. All animals undergoing surgery were anesthetized with ketamine (80 mg/kg, Fort Dodge Animal Health; Fort Dodge, IS) and xylazine (10 mg/kg, The Butler Company; Columbus, OH) before undergoing a laminectomy at the T10 vertebral level and receiving a moderate (150 kdyn) spinal cord contusion using the well-characterized IH impacting device (PSI, Fairfax, VA) (Rabchevsky et al., 2003; Scheff et al., 2003). A 2.5 mm impactor tip was set 8 mm above the spinal cord. After impact, the wounds were irrigated with saline, and the muscle and fascia were reapproximated with absorbable sutures. The skin was closed with surgical staples. The animals had their temperature maintained at a constant level with heating pads during the surgery and post-operative period.
4.2.
Post-injury care
Each rat received 10 ml sterile saline injected subcutaneously and 33.3 mg/kg cefazolin (SoloPak Laboratories; Elk Grove, IL) intramuscularly, immediately post-operation, as well as on post-injury days 1 and 2; additional cefazolin was given for any evidence of hematuria/urinary tract infection. Bladders were manually expressed twice daily using the method of Crede until bladder function returned. Animals had access to food and water ad libitum. The rats were housed under the auspices of the University of Kentucky Animal Care Facility.
4.3.
Experimental design
4.3.1.
5-Day supplementation arm
Male (n = 19) and female (n = 19) rats were assigned to one of three different treatment groups: vehicle (dimethylsulfoxide, DMSO (Sigma; St. Louis, MO)), 4 mg/kg PG, and 8 mg/kg
progesterone (PG (P8783, Sigma; St. Louis, MO)). Following the contusional SCI, animals received PG therapy at 30 min, 6 h and once daily for 5 days. This paradigm recreates the one used by Thomas et al. (1999) and adds an 8 mg/kg group.
4.3.2.
14-Day supplementation arm
Male (n = 18) and female (n = 19) rats were assigned to one of three different treatment groups: vehicle (hydroxypropyl-βcyclodextrin (Sigma; St. Louis, MO)), 8 mg/kg PG, and 16 mg/kg PG. Following SCI, animals received PG therapy at 30 min, 6 h, and once daily for 14 days post-injury.
4.4.
Behavioral analyses
To examine possible effects of progesterone on locomotor recovery, animals were tested and scored using the Basso, Beattie, Bresnahan Locomotor Rating Scale (BBB) (Basso et al., 1996). Animal activity was scored by at least two trained individuals who were blinded to the treatments of each animal. Scoring discrepancies were discussed by the evaluators at the time of testing; averages were taken. BBB scores were generated for each hind limb, with the average of the two limbs (rounded down) tabulated at post-injury days 2, 7, 14, and 21; the rats were assessed pre-injury to assure no baseline deficits. Mean scores were calculated for each treatment group with the differences investigated using analysis of variance (ANOVA) via StatView 4.5.3 statistical package (Abacus Concepts Inc: Berkeley, CA) (Scheff et al., 2002). Significance was set at p < 0.05 for all experiments.
4.5.
Spinal cord harvesting and processing
At day 21 post-injury, animals were overdosed with sodium pentobarbital (Abbott Laboratories; North Chicago, IL) and transcardially perfused with 0.1 M phosphate-buffered saline (PBS, pH 7.4) and 4% paraformaldehyde (Sigma; St. Louis, MO) in PBS. SC was dissected away from surround tissue and bone from T6-L1, as a 3 cm segment. The SC was then additionally fixed in 4% paraformaldehyde for 24 h, washed in PBS for 24 h, then cryoprotected in 20% sucrose/PBS at 4 °C prior to embedding in gum tragacanth (Sigma; St. Louis, MO) and snap frozen in acetone cooled on dry ice. The embedded spinal cords were stored at −80 °C until sectioning. The embedded SC
Fig. 2 – Representative images of the spinal cord damage following a 150 kdyn contusional injury. Image B is the injury epicenter; the other images are the next section, 1 mm distant, cranial (A) and caudal (C) to the epicenter. The bar indicates 1 mm.
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were serially cross-sectioned at 20 μm in a Jung Cryostat set at −25 °C. Every fifth section of 20 μm thickness was placed, sequentially, on slides numbered 1–10; thusly each slide contained sections 1 mm apart. Ten sets of slides, each with about 30 sections, 1 mm apart, were available for morphometric analysis. The slides were stored at − 20 °C until use.
4.6.
Staining and morphometric analysis
The slides were stained with a modified eriochrome cyanine (EC) staining protocol which allowed for differentiation of white matter and grey matter (Rabchevsky et al., 2001). Stereologic image analysis was performed on the EC-stained sections using NIH Image v1.62 (NIH; Betheseda, MD). To minimize differences between animals, the ten sections (10 mm) cranial to the epicenter, and the ten sections caudal to the epicenter were used for analysis. At least two sets of sections were available for review per spinal cord. Length of injury was determined by taking the number of sections with identifiable injury present and multiplying it by 1000 μm (distance between sections). Spared tissue was determined by the presence of preserved architecture with the EC staining of myelin in white matter (WM) and cell body staining in grey matter (GM). The epicenter was defined as the section with the least percentage of spared tissue (Fig. 2). Nearly all the persevered tissue at the epicenter is WM; an inconsequential amount is GM. Therefore, percent spared tissue at the epicenter reflects preserved WM which correlates to the histologic measurements in Thomas et al. The volume assessments were calculated assuming linear gradation and 1 mm distance between section and using the Cavlieri method (Rabchevsky et al., 2001). Total WM volume was obtained by subtracting total GM volume and total injury volume from total volume (Rabchevsky et al., 2000). Predicted total volume, predicted GM volume, and predicted WM volume were determined by averaging the total area, GM area, and WM area in five uninjured sections rostral and caudal to the injury; these results were used to extrapolate the pre-injury areas in the injured SC segments (Rabchevsky et al., 2001). Because females have smaller SC than males, percent spared tissue was calculated to allow for gender comparisons. These were calculated by dividing actual volumes by predicted volumes.
Acknowledgment The research was funded in part by the Kentucky Spinal Cord and Head Injury Trust #05A.
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