Accumulation of connective tissue growth factor⁺ cells during the early phase of rat traumatic brain injury

Glial scar formation is a common histopathological feature of traumatic brain injury (TBI). It persists for long periods and acts as barrier not only to axon regeneration but also to inflammatory cells in a manner that protects healthy tissue from nearby areas of intense inflammation [1]. Reactive astrocytosis is the key contributes to glial scar formation [2]. Induced by stimuli from lesions, astrocytes enter into the injured zone, are activated, and play a major role in the response to TBI. In central nervous system (CNS) injuries, activated astrocytes secrete many cytokines, among which transforming growth factor (TGF)-β is regarded as one of the most potent cytokines, it can be selectively upregulated in astrocytes and contribute to scar formation after injury through inducing secretion of another “downstream mediator”, the cytokine connective tissue growth factor (CTGF) [3, 4].

CTGF is a secreted peptide encoded by an immediate early growth responsive gene that has been shown to be a downstream mediator of TGF-β1 action, induces mitogenesis, chemotaxis, and cell matrix induction of fibroblasts [5]. Due to these properties, CTGF plays important roles in the regulation of scar formation, wound healing, cell migration, proliferation and extracellular matrix [4, 6]. A series of studies has also demonstrated that CTGF over-expression correlates with many fibrotic and inflammation-associated diseases, such as fibrotic skin disease, atherosclerosis and inflammatory bowel disease [7‐9].

Upregulation of CTGF has been observed in a variety of nervous system-related disorders. In Alzheimer’s disease, CTGF was observed to overexpress in perivascular astrocytes and in astrocytes associated with plaques, indicating the role for CTGF in the process of chronic neurodegeneration [10]. In amyotrophic lateral sclerosis, CTGF was dramatically increased in reactive astrocytes of the ventral horn, supporting a role for CTGF in the molecular mechanisms underlying astrogliosis [11]. Although the expression of CTGF has been reported in human TBI, mice spinal cord injury, and rat kainic acid-induced brain injury, altered CTGF expression following TBI models are not completely clear. Therefore, in the current study, we have investigated the spatiotemporal expression of CTGF following an open-skull weight-drop-induced TBI in rat brains.

A reproducible local lesion is formed on open-skull weight-drop injury in the ipsilateral cortex. The development of the lesion following TBI was analyzed using H&E staining has been described previously [13]. In brief, selective neuronal loss and necrotic loci condensed at the edge of impact site were observed 12 h postinjury (p.i.). Significant leukocyte infiltration and hemorrhage were already observed on day 1. At day 4 post-TBI, a lesioned cavity together with its surrounding perilesional tissue loss was formed under the impact areas. At the lesional areas, most tissue was lost, no neurons remained, and leukocyte infiltration together with hemorrhage appeared. In the perilesional areas, selective neuron loss with leukocyte infiltration was observed. In the subcortical white matter, no morphological changes were observed.

The expression of CTGF post-TBI was studied using immunohistochemistry. In normal adult rat brain, CTGF immunoreactivity was rarely observed in the cortex and meningeal (0.20 ± 0.194 per high-power field, Figures 1A and 2), only a few CTGF immunoreactivity was demonstrated in small vessels (Figure 1A, box indicated). Furthermore, almost all ependymal cells showed CTGF⁺ (Figure 1B). After TBI, no visible change in CTGF immunoreactivity was detected in the contralateral hemisphere (data not shown). In the injured (ipsilateral) sides, we found that CTGF expression in neurons did not change significantly, but the accumulation of non-neuron CTGF⁺ cells was observed in the lesion and perilesional areas (Figure 1C, arrows indicated). We then quantified the non-neuron CTGF⁺ cells in lesional areas. CTGF⁺ neurons were not counted. After TBI, a slight increase in CTGF⁺ non-neuron cells was observed confined to the lesion as early as 6 h p.i. (0.50 ± 0.497 per high-power field, P > 0.05, Figures 1D and 2). The accumulation of CTGF⁺ non-neuron cells increased over time (Figures 1E, F and 2). The accumulation of CTGF⁺ non-neuron cells became significant at 72-h post-TBI (17.40 ± 1.503 per high-power field, P < 0.01, Figures 1G and 2) and reached the maximum at 96-h post-TBI (74.20 ± 7.248, P < 0.01, Figures 1H and 2) during our observed time period.

The accumulation of CTGF⁺ non-neuron cells was mainly in perilesional areas but not in remote areas of the ipsilateral and contralateral hemispheres. At the lesioned site, most of the CTGF⁺ non-neuron cells showed activated astrocytes phenotypes with typical stellate morphologic characteristics or belt-shaped, grouped pattern (Figure 1G, box indicated). CTGF immunoreactivity was also detected in endothelial cells (Figure 1F, arrows indicated), but no significant differences were observed between injured and control brains.

Here we analyzed the accumulation of CTGF⁺ non-neuron cells in rat brain of weight-drop contusion following TBI. Significant CTGF⁺ non-neuron cell accumulation was detected 72-h p.i. and increased markedly. The CTGF⁺ non-neuron cells mainly detected in perilesioned areas where CTGF accumulation various in different time course. Furthermore, most CTGF⁺ non-neuron cells showed activated astrocyte phenotypes with typical stellate morphologic characteristics.

Nowadays, increasingly studies are focusing on the recovery stage after neural tissue injury [14], through which more pro-recovering molecules are discovered [15]. CTGF is investigated as a secreted cell matrix-inducing peptide involved in both tissue regeneration mechanisms [3]. In normal rat brains, CTGF expression can be found in glia-like cells, neurons, endothelial cells and ependymal cells [6]. In the CNS, increased expression of CTGF was induced under certain pathological conditions, such as spinal cord injury and cerebral infarction [6, 16]. It was demonstrated that in kainic acid-induced brain injury, significantly increasing of CTGF mRNA expression was limited in the ipsilateral hippocampus, no CTGF protein expression was observed in non-lesioned hippocampus [16]. However, in the current investigation, using an open-skull weight-drop injury, which is considered to cause less learning and memory deficits and produce less degenerating neurons compared with kainic acid-induced brain injury model [17], the accumulation of non-neuron CTGF⁺ cells was not only in the direct vicinity of the lesion core, but also in cells with astrocyte phenotypes which located on non-lesioned areas.

Accumulation of CTGF⁺ astrocytes is a well-established response to traumatic brain injury, which contributes to wound healing and glial scar formation, and is beneficial for TBI recovery [16]. Reactive CTGF⁺ cells with astrocyte phenotypes accumulated rapidly after TBI. In 6-h p.i., CTGF⁺ cells with astrocyte phenotypes accumulated in the direct vicinity of the lesion core in the cortex, while as early as 6 h p.i., CTGF⁺ cells with parenchymal astrocyte phenotypes was detected in the non-lesioned areas. These results suggest a participation of CTGF in the pathological features within the astrocytic compartment. Glial/collagen scarring is composed of dense packed reactive astrocytes and matrix protein deposition sealed by formation of a new basal lamina. Hence, the restricted expression and accumulation of CTGF in TBI adds convincing evidence for the participation of CTGF in the early astrocytic CNS injury response as a key molecule contributing to TBI recovery.

We have found an early accumulation of CTGF⁺ cells in open-skull weight-drop-induced TBI. Our study suggests that CTGF expression might define a subtype of activated astrocytes with a specific role in the tissue-repair processes following TBI.