Overexpression of kynurenic acid and 3-hydroxyanthranilic acid after rat traumatic brain injury

Abstract

Using an immunohistochemical technique, we have studied the distribution of kynuneric acid (KYNA) and 3-hydroxyanthranilic acid (3-HAA) in a rat brain injury model (trauma). The study was carried out inducing a cerebral ablation of the frontal motor cortex. Two mouse monoclonal specific antibodies previously developed by our group directed against KYNA and 3-HAA were used. In control animals (sham-operated), the expression of both KYNA and 3-HAA was not observed. In animals in which the ablation was performed, the highest number of immunoreactive cells containing KYNA or 3-HAA was observed in the region surrounding the lesion and the number of these cells decreased moving away from the lesion. KYNA and 3-HAA were also observed in the white matter (ipsilateral side) located close to the injured region and in some cells placed in the white matter of the contralateral side. The distribution of KYNA and 3-HAA perfectly matched with the peripheral injured regions. The results found were identical independently of the perfusion date of animals (17, 30 or 54 days after brain injury). For the first time, the presence of KYNA and 3-HAA has been described in a rat trauma model. Moreover, by using a double immunocytochemistry protocol, it has been demonstrated that both metabolites were located in astrocytes. The findings observed suggest that, in cerebral trauma, KYNA and 3-HAA are involved in tissue damage and that these compounds could act, respectively, as a neuroprotector and a neurotoxic. This means that, in trauma, a counterbalance occurs and that a regulation of the indoleamine 2,3 dioxygenase (IDO) pathway could be required after a brain injury in order to decrease the deleterious effects of ending metabolites (the neurotoxic picolinic acid). Moreover, the localization of KYNA and 3-HAA in the contralateral side of the lesion suggests that the IDO pathway is also involved in the sprouting and pathfinding that follows a traumatic brain injury.

Figure 1.

Low-power magnifications of brains after fixation. A) Sham operated animal; B) operated animal; the arrow indicates the lesion conducted on the motor cortex.

Figure 2.

Immunoreactivity for KYNA. Ipsilateral side of the ablated motor cortex (A-D). A) Note the expression of KYNA in the region close to the lesion; the number of immunoreactive cells decreased moving away from the injured region. The region delimited by a rectangle is shown, at a higher magnification, in panel C. B) Picture of another region showing the immunoreactivity all around the lesion; C) The rectangle in the upper right corner is a higher magnification of the small left rectangle. D) High power magnification image, note the morphology of the cells containing KYNA. Small arrow D, dorsal orientation.

Figure 3.

Photographs taken from the region around the lesion. Double-labelling of astrocytes containing GFAP and KYNA (A, B) or GFAP and 3-HAA (C, D). The immunolabelling for GFAP appears in blue (chloronapthol) and that of KYNA or 3-HAA in brown (diaminobenzidine). Note the blue staining in the astrocyte projections (arrowheads) and the brown staining in the cell bodies. Small arrow D, dorsal orientation.

Figure 4.

Immunoreactivity for 3-HAA. Ipsilateral side of the motor cortex. A) Note the expression of 3-HAA in the region close to the lesion; the number of immunoreactive cells decreased moving away from the injured region; the region delimited by the upper rectangle is shown at higher magnification in panel B, and that of the lower rectangle in panel C. B) High-power magnification of the region delimited by the left rectangle is shown in panel D; right rectangle: a detail of another region showing immunoreactive cells. D) Rectangle, detail of cells containing 3-HAA. Small arrow V, ventral orientation.

Figure 5.

Immunoreactivity for IBA-1. A) High density of immunoreactive cells around the injured region. B) High-power magnification of the region delimited by the rectangle in A. C) Representative image of the labeling with IBA-1 observed in the contralateral side of the motor cortex or in sham-operated animals; compare the immunolabelling appearing in A and B. D) Control: absence of labelling when the first antibody was omitted in the immunocytochemical technique. Small arrow D, dorsal orientation.