Early and specific expression of Monocyte Chemoattractant Protein-1 in the thalamus induced by cortical injury¹

Abstract

For many years it has been known that retrograde degeneration of thalamic neurons occurs following damage to the cerebral cortex, however, the molecular mechanisms which control this process are unknown. Recent studies have demonstrated microglial activation in thalamic nuclei well before the onset of retrograde neuronal cell death. Activated monocytes and microglia synthesize factors detrimental to neuronal survival as well as phagocytose damaged and dying neurons. Our previous studies demonstrated that monocyte chemoattractant protein-1 (MCP-1), a β chemokine which attracts cells of monocytic origin to sites of injury, is rapidly expressed in the brain following visual cortical lesions. The present study examined the expression of MCP-1 messenger RNA and protein in the thalamus following a visual cortical lesion. Aspiration lesions of visual cortex were made in adult mice. At specific times after lesion, brains were harvested and dissected into specific regions. MCP-1 message as detected using northern analysis was absent in uninjured brain, but was elevated in the ipsilateral thalamus as rapidly as 1 h following the lesion. In situ hybridization localized MCP-1 message to subpial glial cells of the lateral geniculate nucleus (LGN) of the ipsilateral thalamus after injury. ELISA showed that MCP-1 protein levels were significantly elevated in the ipsilateral thalamus at 6 h, peaked at 12 h, and remained above baseline levels for at least 1 week post lesion. In addition, anti-GFAP staining demonstrated activated astrocytes localized to the ipsilateral LGN at 24 and 72 h after injury. The early expression and regional localization of MCP-1 mRNA and protein strongly suggest that MCP-1 is a critical molecule in the regulation of thalamic retrograde neuronal degeneration.

Introduction

Injury to the neocortex results in axotomy-induced retrograde degeneration of thalamic neurons which induces cell death several days later [16], [23], [33], [46]. Recent work by Agarwala and Kalil [1] indicates that dying neuronal subpopulations follow one of two pathways following focal cortical lesion of the visual cortex. Within the first 3 days after injury, a small percentage of thalamic neurons die by necrosis while the vast majority of neurons show no morphological change. However, in the subsequent 4 days, massive neuronal cell death occurs by apoptosis. The general pattern of minimal neuronal cell death with limited morphological change followed by a precipitous period of massive cell death also occurs following other types of CNS injury [5]. The mechanisms regulating delayed neuronal death remain obscure. In order to eventually develop treatments to stimulate functional recovery after brain injury, it is crucially important to understand the mechanism(s) that initiate delayed neuronal death. Retrograde neuronal degeneration following cortical injury provides a defined population of neurons destined to die within a specific period of time.

Cells of the monocyte/macrophage lineage including microglia play a critical role in a variety of tissue remodeling events. Macrophages are essential for the recovery and repair of injured peripheral tissues. However, activated macrophages also produce factors which are detrimental to cell survival presenting a potential dual role for these monocyte derived cells. Peripheral tissues have a much greater regenerative potential than the CNS and thus can more easily overcome secondary damage from activated monocytic cells. For reasons not well understood, the CNS is much more limited in its ability to recover and regenerate connections after injury. Recent studies have shown that cells of monocytic origin are important in the pathological processes that lead to neuronal cell death. Activated microglia contribute to neuron death when they phagocytose damaged but viable axotomized retinal ganglion cells [52]. Significantly, suppression of microglial activation with macrophage inhibitory tripeptide (Thr²⁸⁹–Lys–Pro²⁹¹) results in survival of neurons, with the possibility of functional recovery indicated by axonal outgrowth. In addition, blockage of macrophage recruitment, attenuation of macrophage response or depletion of macrophages after CNS trauma also promotes neuronal survival and improves subsequent neurologic function [8], [23], [41], [42].

The general response to injury and inflammation in the CNS is slower than in peripheral tissues and moreover, is primarily mononuclear in nature [38], [39]. There has been considerable speculation on the reasons for the monocyte-specific injury response in the CNS, however, molecular mechanisms responsible for the migration and activation of these inflammatory cells to the region surrounding neuronal perikarya are unknown. One mechanism may be selective chemokine expression. Chemokines (a contraction of chemoattractant and cytokine) are small, inducible, secreted members of the cytokine family acting as attractants for cells involved in inflammatory processes. Chemokines are produced by a variety of cell types and have multiple targets, however, monocytes/macrophages appear to be a common source and target for many chemokines [47]. Chemokines are classified into two main subfamilies: the CXC or α chemokines which primarily recruit neutrophils and the CC or β chemokines which primarily recruit mononuclear inflammatory cells [37], [47]. Locally produced chemotactic molecules are thought to be responsible for recruitment of inflammatory cells into the CNS parenchyma [48], [51].

Monocyte chemoattractant protein-1 (MCP-1) is a member of the β chemokine subfamily. In peripheral tissues, MCP-1 is critical for recruitment of inflammatory cells of monocytic origin after injury or inflammation [18]. While recent investigations show that MCP-1 is not expressed in normal CNS [7], [26], MCP-1 is upregulated during CNS inflammation [7], [21], [26]. Activated astrocytes and cells of monocytic origin have been shown to produce MCP-1 in vivo [12], [24], [44]. The present study was designed to determine the temporal and regional distribution of MCP-1 mRNA and protein following a visual cortical lesion. Results of these studies demonstrate the presence of MCP-1 mRNA and protein in the thalamus prior to any sign of retrograde neuronal degeneration.