Inhibition of Transforming Growth Factor-β Attenuates Brain Injury and Neurological Deficits in a Rat Model of Germinal Matrix Hemorrhage
Background and Purpose—Transforming growth factor-β (TGF-β) overproduction and activation of the TGF-β pathway are associated with the development of brain injury following germinal matrix hemorrhage (GMH) in premature infants. We examined the effects of GMH on the level of TGF-β1 in a novel rat collagenase-induced GMH model and determined the effect of inhibition of the TGF receptor I.
Methods—In total, 92 seven-day old (P7) rats were used. Time-dependent effects of GMH on the level of TGF-β1 and TGF receptor I were evaluated by Western blot. A TGF receptor I inhibitor (SD208) was administered daily for 3 days, starting either 1 hour or 3 days after GMH induction. The effects of GMH and SD208 on the TGF-β pathway were evaluated by Western blot at day 3. The effects of GMH and SD208 on cognitive and motor function were also assessed. The effects of TGF receptor I inhibition by SD208 on GMH-induced brain injury and underlying molecular pathways were investigated by Western blot, immunofluorescence, and morphology studies 24 days after GMH.
Results—GMH induced significant delay in development, caused impairment in both cognitive and motor functions, and resulted in brain atrophy in rat subjects. GMH also caused deposition of both vitronectin (an extracellular matrix protein) and glial fibrillary acidic protein in perilesion areas, associated with development of hydrocephalus. SD208 ameliorated GMH-induced developmental delay, improved cognitive and motor functions, and attenuated body weight loss. SD208 also decreased vitronectin and glial fibrillary acidic protein deposition and decreased GMH-induced brain injury.
Conclusions—Increased level of TGF-β1 and activation of the TGF-β pathway associate with the development of brain injury after GMH. SD208 inhibits GMH-induced activation of the TGF-β pathway and leads to an improved developmental profile, partial recovery of cognitive and motor functions, and attenuation of GMH-induced brain atrophy and hydrocephalus.
Germinal matrix hemorrhage (GMH) is one of the most common and devastating cerebrovascular events that affects premature infants.1 It occurs in up to 20% of infants delivered at <32 weeks’ gestation. In the United States, >12 000 premature infants develop GMH every year.2 Infants with GMH are at risk of posthemorrhagic hydrocephalus and cerebral palsy, potentially leading to mental retardation and lifelong developmental disabilities.3 Despite ongoing research on new therapeutic strategies, intensive rehabilitation remains the only option for improvement of quality of life for these children.
Transforming growth factor-β (TGF-β) is a highly conserved protein with 3 isoforms. Although TGF-β2 and TGF-β3 are constitutively expressed in the brain, there is a dramatic increase in TGF-β1 level in the brain after a hemorrhagic event.4–6 The effects of TGF-βs are mediated by 3 receptors.7 The role of the type III receptor (TGFR-III) is unclear, although a report suggests that it may function to present TGF-β to the type I and II receptors.8 The roles of TGFR-I and TGFR-II have been more clearly elucidated. Constitutively phosphorylated TGFR-II binds to TGF-β. The binding between TGFR-II and TGF-β induces phosphorylation of TGFR-I.9 On phosphorylation, activated TGFR-I phosphorylates the TGF-β pathway signaling molecules SMAD2 and SMAD3 (SMAD2/3),10 which act as transcription factors in the nucleus.
An association between the increased level of the TGF-β1 after injury and development of hydrocephalus has been observed in both animal models and premature newborns.5 Moreover, overexpression of TGF-β1 in genetically modified animals and direct injection of TGF-β1 into animal brains cause the development of hydrocephalus.11,12 It has become apparent that TGF-β plays a pivotal role in the modulation of extracellular matrix (ECM) proteins, acting to regulate the synthesis and degradation of their components.13,14 ECM protein deposition impairs absorption of cerebral spinal fluid and is an important contributor to the development of the posthemorrhagic hydrocephalus.15
In this study, we examined the effect of collagenase-induced GMH in rats on level of TGF-β1 and on the state of activation of TGFR-I. We hypothesize that increased level of TGF-β1 results in accumulation of ECM proteins and glial fibrillary acidic protein (GFAP), associated with hydrocephalus, brain atrophy, and significant neurological deficits. We also postulate that inhibition of the TGF-β pathway via a novel TGFR-I inhibitor ameliorates GMH-induced brain damage and neurological deficits.
Materials and Methods
All experiments were approved by the Loma Linda University Institutional Animal Care and Use Committee.
GMH was induced as described.16 Pregnant Sprague–Dawley rats were purchased from Harlan Laboratories (Indianapolis, IN). Ninety-two P7 rat pups were then randomly divided into the sham-operated (n=20), collagenase-injected vehicle-treated (n=44), collagenase-injected SD208-treated (n=28) animals. Pups of both sexes were used. An aseptic technique was used. For GMH induction, animals were anesthetized with 3% isoflurane and placed onto stereotaxic frame. Isoflurane concentration was then reduced to 2%. The scalp area was sterilized, and bregma was exposed. Using bregma as a reference point, the following stereotactic coordinates were measured: 1.8 (rostral) and 1.5 mm (lateral). A burr hole (1 mm) was drilled. A 27-gauge needle was inserted at a rate of 1 mm/min at the depth of 2.8 mm from the dura. Using a microinfusion pump (Harvard Apparatus, Holliston, MA), 0.3 U of clostridial collagenase VII-S (Sigma, St Louis, MO) in 0.5 μL was infused through the Hamilton syringe. The needle remained in place for an additional 10 minutes after injection to prevent back leakage. After the needle was removed, the burr hole was sealed with bone wax and the incision suture closed, and the animals were allowed to recover on a 37°C heated blanket. On recovering from anesthesia, the animals were returned to their dams. Sham operation consisted of needle insertion alone without collagenase infusion.
SD208 was purchased from Tocris Bioscience and injected intraperitoneally. Two concentrations of SD208 were tested: 20 mg/kg (low concentration) and 60 mg/kg (high concentration).17 Untreated animals received an injection of equal volume of vehicle (0.1% of dimethyl sulfoxide). Two treatment strategies were used: an acute treatment, in which we injected SD208 daily for 3 days starting at 1 hour after induced GMH, and a delayed treatment, in which we injected SD208 daily for 3 days starting at day 3 after induced GMH.
The effects of GMH and treatment with SD208 on the development of animals were evaluated using body righting and negative geotaxis tests. Tests were performed in a blinded fashion daily through day 7.16 We also tested the effects of both GMH and SD208 treatment on cognitive function using T-maze, water maze, and motor function using foot faults and neurodeficit scales16 in a blinded fashion between days 21 and 24 (online-only Data Supplement).
The effects of GMH on TGFR-I and TGF-β1 level were evaluated at 3, 6, 12, 27, and 72 hours after GMH by Western blot according to the manufacturer’s recommendations.18 The effects of SD208 on the GMH-induced activation of the TGF pathway were evaluated by Western blot on day 3, 1 hour after the last drug injection (online-only Data Supplement).
Immunohistochemistry and Brain Injury Evaluation
Brain atrophy was evaluated by calculating brains’ body weight, changes in total brain tissue loss, and brain loss in cortical and subcortical areas at day 24.16 The effects of SD208 on GMH-induced accumulation of vitronectin and GFAP were evaluated at day 10 for GFAP and at day 24 for vitronectin16 as previously described (online-only Data Supplement).
Data were analyzed by 1-way ANOVA followed by Tukey post-test. A P value of <0.05 was considered statistically significant. Significance in differences of the water maze test was estimated using a 95% confidence interval overlap measure. All statistical analyses were performed using SigmaStat. Values are expressed as mean±SD.
Effect of GMH and SD208 on Survival of Animals
Neither collagenase-induced GMH nor administration of SD208 caused significant mortality in this study. Only 1 GMH vehicle-treated animal died overnight after GMH induction.
Effect of GMH and SD208 on TGF Pathway
Although GMH had no significant effect on TGFR-I level (Figure 1A), a significant increase in TGF-β1 level at 3 and 6 hours after hemorrhage was observed (*P<0.05 versus sham, Figure 1B). Neither GMH nor SD208 had a noticeable effect on the level of the downstream TGFR-I molecules SMAD 2/3 (Figure 2A). GMH increased phosphorylation of SMAD 2/3 and SD208 decreased SMAD 2/3 phosphorylation (*P<0.05 versus sham; #P<0.05 versus vehicle; Figure 2B). High-dose SD208 significantly decreased the ratio of phosphorylated to nonphosphorylated SMAD 2/3 compared with untreated animals (*P<0.05 versus sham; #P<0.05 versus vehicle; Figure 2C).
Effect of SD208 on GMH-Induced Neurological Deficits
At days 1 and 2, we observed significant developmental delay (evaluated by both body righting and negative geotaxis tests) in all vehicle-treated GMH rats compared with sham-operated animals (*P<0.05 versus sham; Figure 3A and 3B). High-dose SD208 ameliorated GMH-induced neurological deficits (#P<0.05 versus vehicle; Figure 3A and 3B); the low dose was ineffective (data not shown). GMH-induced developmental delay resulted in long-term adverse effects on both cognitive and motor function compared with sham-operated animals (*P<0.05 versus sham; Figure 4A–4D). Because only the high dose of SD208 was effective in the short term, this dose was also used for our long-term study.
Acute treatment improved cognitive and motor functions evaluated by T-maze and water maze as well as by neuroscore and foot fault tests. Delayed treatment significantly improved the cognitive function of treated animals and improved motor functions evaluated by the foot fault test (P<0.05 delayed versus vehicle; Figure 4A). In addition, we showed a strong tendency toward improvement of motor function evaluated by the foot fault test.
Effect of SD208 on GMH-Induced Weight Loss, Brain Loss, and Ventriculomegaly
Twenty-four days after GMH, we observed significant weight loss in GMH animals compared with sham-operated animals (from 119.5±4.6 to 90.5±7.8 g; sham versus GMH animals, respectively). Both acute and delayed treatments with SD208 had a tendency to attenuate GMH-induced weight loss (mean weights, 107.4±3.7 for the acute treatment group and 114.6±6.1 for the delayed treatment group), but this did not reach significance (Figure in the online-only Data Supplement).
We observed a significant decrease of the brain weight in the rats with induced GMH compared with sham-operated animals. High-dose SD208 ameliorated GMH-induced brain atrophy in both the acute treatment group and delayed treatment group (*P<0.05 versus sham; Figure 5A).
Compared with sham animals, GMH caused significant brain tissue loss in both cortical (ipsilateral hemisphere, 83.12±2.74 versus 69.91±3.86 mm3, sham and GMH animals, respectively) and subcortical areas (ipsilateral hemisphere, 72.35±3.96 versus 47.86±1.89 mm3, sham and GMH animals, respectively; contralateral hemisphere, 75.85±1.91 versus 59.28±3.14 mm3, sham and GMH animals, respectively). Total brain tissue loss was also observed in GMH compared with sham animals (ipsilateral hemisphere, 150.25±6.56 versus 116.01±5.24 mm3, sham and GMH animals, respectively; contralateral hemisphere, 157.89±2.84 versus 143.49±5.83 mm3, sham and GMH animals, respectively). Both the acute and delayed treatments significantly attenuated GMH-induced brain tissue loss in the subcortical area (ipsilateral hemisphere tissue loss, 47.86±1.89 versus 60.16±3.11 mm3 in acute and delayed treatment group, respectively; contralateral hemisphere tissue loss, 59.28±3.14 versus 72.23±3.05 and 71.48±1.56 mm3 in acute and delayed treatments, respectively) and showed a strong tendency to decrease brain loss in another brain areas (#P<0.05 versus vehicle; Figure 5B).
Animals with induced GMH showed increased contralateral and ipsilateral ventricular volumes with contralateral ventricular volume changes of 1.93±0.43 versus 27.13±10.97 mm3 and ipsilateral volume changes of 2.83±1.15 versus 57.86±13.23 mm3 for sham versus GMH groups, respectively (Figure 5C and 5D). SD208 ameliorated GMH-induced ventriculomegaly with contralateral ventricular volume changes of 7.13±10.97 versus 4.35±0.92 mm3 (vehicle and acute treatment, respectively) and ipsilateral ventricular volume changes of 57.86±13.23 versus 27.82±3.92 mm3 (vehicle and delayed treatment, respectively; #P<0.05 versus vehicle; Figure 5D).
Effect of SD208 on GMH-Induced Vitronectin and GFAP Accumulation
We have previously shown that in induced GMH, GFAP level peaks on postevent day 10 and vitronectin level peaks on postevent day 24.16 In this study, we tested the effects of acute treatment with SD208 on those time points. We demonstrated that TGF-β pathway inhibition by SD208 significantly decreased the level of GFAP (Figure 6A). On day 24, SD208 decreased GMH-associated vitronectin level as measured by Western blot (Figure 6B).
GMH is a common consequence of premature newborns. In this study, we examined the effects of collagenase-induced GMH in rats on the activation state of the TGF-β pathway. In addition, we explored the effects of a potent TGFR-I inhibitor on GMH-induced brain injury and associated neurological consequences. The most significant of these neurological sequelae include posthemorrhagic hydrocephalus and brain atrophy which may potentially lead to developmental delay and mental retardation.19 Hemorrhagic brain injury causes a dramatic increase in TGF-β1 level in the brain, which can subsequently lead to the development of hydrocephalus.4–6 Overexpression of TGF-β1 in genetically modified animals and direct injection of the TGF-β1 into the brain of animals also have been shown to cause development of hydrocephalus.11,12
Previously, we described a novel collagenase model of GMH that is able to mimic the motor deficits and ventricular dilation seen in human preterm newborns.16 The molecular pathways leading to these events remain to be elucidated, and this is one of the goals of the present study. In this study, we observed a significant increase of TGF-β1 level after induced GMH via collagenase injection. This corroborates with other studies demonstrating increased TGF-β1 level in the central nervous system in response to a hemorrhagic event.4–6 We postulate that activation of the TGF-β pathway is at least partly responsible for the development of brain injury and neurological deficits following GMH.
We hypothesize that TGF-β pathway inhibition can ameliorate the devastating effects of GMH.16 We used a novel, potent inhibitor of TGFR-I (SD208) because TGFR-I is a last step in the activation of the TGF receptor complex. SD208 displays 100-fold selectively to binding TGFR-I over TGFR-II. After systemic application, SD208 limits the bioavailability of TGF-β in brain by decreasing SMAD2 and 3 phosphorylation.20–22 A therapeutic approach involving inhibition of TGF-β downstream signaling molecules may hold promise for future treatment modalities. We have demonstrated that systemic administration of SD208 decreases phosphorylation of SMADs in the collagenase model of GMH. The observed effect was dose dependent in that only high doses of TGFR-I inhibitor caused a significant decrease of the phosphorylated to nonphosphorylated SMAD 2/3 ratio.
We tested the effects of TGFR-I inhibition on the development of neurological deficits both at early and late time points in juvenile rats. For evaluation of the developmental profile in neonatal animals, we used negative geotropism and righting-reflex tests. Both of these tests have been previously described for early reflex locomotor testing in rats.23,24 In agreement with previous publications, we observed significant delays in the development of GMH animals, with spontaneous resolution of neurological deficits on day 3.16,25 Inhibition of TGFR-I had a dose-dependent effect on amelioration of GMH-induced neurological deficits in the short-term study.
Developmental delay predicts an impairment of neurological function both in premature born human infants and in animal models.26,27 In the long-term study, we also tested whether treatment with SD208 would ameliorate GMH-induced weight loss, which has been previously described.16 We compared 2 different treatment strategies: acute (starting 1 hour after GMH) and delayed (starting 3 days after GMH).28 In agreement with our previous publication, we observed significant whole-body weight loss in GMH but not sham-operated animals. Both acute and delayed treatments showed a strong tendency to decrease whole-body weight loss. However, the difference between treated and untreated animals was not significant.
We investigated the relationship between the beneficial effects of TGFR-I inhibition observed in the short-term study on motor function and in the long-term study on spatial memory. The effects of TGF pathway inhibition on motor function in juvenile animals were investigated by the neurodeficit scales neuroscore and the foot fault test.16,29 GMH caused significant neurological deficits detectable by both of these tests. Both acute and delayed treatment ameliorated GMH-induced impairment of motor function. In addition, we confirmed that GMH caused spatial learning and memory deficits evaluated by water maze and T-maze tests, respectively.16 In the water maze, no differences during the cued (visible escape platform) learning ability between experimental groups were observed. During the spatial trials (submerged/hidden platform), however, GMH animals performed significantly worse. In addition, GMH animals demonstrated a loss of working memory as evaluated by the T-maze. Both acute and delayed treatments ameliorated GMH-induced learning and memory deficits. Although memory loss is considered the most prominent symptom of hydrocephalus and brain atrophy,30–32 there is an indication that TGF-β1 can cause significant disturbance in the learning process without changes in ventricular size.33 We evaluated brain atrophy by comparison of brain to body ratio between treated and untreated animals. We also used volumetric analyses for calculation of brain tissue loss and ventricular dilation.16 We demonstrated that TGF-β pathway inhibition results in amelioration of brain atrophy and preservation of brain tissue, as well as decreased GMH-induced ventriculomegaly. Posthemorrhagic hydrocephalus results from dysregulation of cerebral spinal fluid production and reabsorption of ECM proteins. A pathological hallmark of hydrocephalus is overproduction and extensive deposition of these proteins.34 TGF-β stimulates the synthesis of ECM proteins and results in increased ECM protein accumulation.35,36 ECM protein degradation is also blocked by inhibition of the synthesis of proteases and increased synthesis of protease inhibitors.37,38 Previously, we demonstrated that collagenase-induced GMH caused significant upregulation of vitronectin at day 17 and 24 after GMH.16 Differences in GFAP levels between sham-operated and GMH animals reached statistical significance on day 10.16 We evaluated the effect of TGF pathway inhibition on the level of vitronectin and GFAP during days when level was maximal: day 24 for vitronectin and day 10 for GFAP. We demonstrated that TGF inhibition significantly decreased GMH-induced levels of both vitronectin and GFAP proteins. We did not evaluate the effect of TGF-β pathway inhibition on cerebral spinal fluid absorption directly, and this will remain a goal for future studies.
In conclusion, GMH increased the level of TGF-β1 and activated the TGF-β pathway in brain tissue. The activation of TGF-β pathway contributed to the development of brain injury after GMH. Inhibition of GMH-induced activation of the TGF-β pathway led to significant cognitive and motor improvement and attenuation of GMH-induced brain atrophy and development of hydrocephalus.
This study is partially supported by NIH NINDS 078755 to J.H.Z.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.113.003754/-/DC1.
- Received October 10, 2013.
- Revision received November 22, 2013.
- Accepted November 26, 2013.
- © 2014 American Heart Association, Inc.
- 1.↵The vermont-oxford trials network: very low birth weight outcomes for 1990. Investigators of the vermont-oxford trials network database project. Pediatrics. 1993;91:540–545.
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