The central nervous system is built from two broad categories of cells, neurons and glial cells which include the astrocytes, oligodendrocytes, ependymal cells and microglia. For a long time glial cells were considered to have a limited role in supporting neurons. Now they are recognized as key players in virtually all aspects of brain function in all vertebrates.1-3 Cell bodies of astrocytes are juxtaposed between neurons and the capillary endothelium to form the blood-brain barrier (BBB). Given these cytoarchitectural features, they are the first cells of the brain parenchyma to encounter foreign molecules crossing this barrier.4 For this they have also a neuroprotective action from cytotoxic action of heavy metals.5 Cadmium (Cd) is a heavy metal with a long biological half-life of ~30 years with a high potential toxicity. Unfortunately this metal is increasingly in the environment as the result of industrial and agricultural practices,6 thereby entering the food chain.7 It is known that an acute exposure to Cd produces toxicity to the lungs, testes and brain, while a chronic exposure to this metal determines renal dysfunction, anaemia and osteoporosis.8-11 Numerous studies provide evidence that Cd induces neuronal toxicity and damage to the brain.12,13 The brain accumulates metals that are often localized in the astroglia.14 In mammals some studies have been carried out on the toxic effect of heavy metals on glia, for this it has been possible to observe that metals like Cd, Tl, Zn and Pb induce morphological changes in the populations of glial cells.15,16 Although the complexity of the cellular and biochemical actions of cadmium has been discussed in a number of studies, the underlying mechanisms are not completely understood. It is not clear whether all the different populations of brain cells (i.e., neurons, glial cells, and endothelial cells) are equally sensitive to this metal. The scope of the present study has been to observe the possible morphological changes induced by CdCl2 on the astroglia of the lizard Podarcis siculus. Among vertebrates, the class Reptilia has been relatively understudied in the field of ecotoxicology, although it is considered to be suitable as bioindicators of chemical exposure due to their persistence in a variety of habitats.17-21 The present study was performed by a morphological analysis of the brain at light microscope and by immunodetection of the glial fibrillary acidic protein (GFAP) in lizards exposed to an acute intraperitoneal treatment with CdCl2. GFAP is the main component of glial filaments expressed in the astroglial cells and it is accepted as a reliable specific molecular marker for these cells.22 GFAP shows a considerable stability in its molecular and antigenic characteristics across vertebrate phylogeny and a clear cross reactivity to the anti-mammalian GFAP also in reptiles.23-27
Materials and Methods
The study was performed on 30 adult females of Podarcis siculus, captured near Naples (Italy) and kept under controlled conditions of light and temperature with food and water ad libitum. A group of 15 specimens received intraperitoneally (i.p.) a single injection (2 mg/kg-BW) of cadmium chloride (Sigma-Aldrich, St. Louis, MO, USA). This dose was chosen according to previous reports.18,19,28 Another group of 15 specimens was used as control by the i.p. injection of physiological solution. Experiments were performed in accordance with the Guideline for Animal Experimentation of the Italian Department of Health under the supervision of a veterinarian, and organised to minimize stress and the number of lizards used. The animals were sacrificed at 2, 7 and 16 days from treatments. The brains were immediately removed and processed.
Histology and immunohistochemistry
The brains of control and treated lizards were fixed in Bouin’s solution for 48 h at room temperature and then dehydrated and enclosed in paraffin. Serial sections of 6 µm were processed by Hematoxylin/Eosin (H&E) and Cresyl/Violet stains for the general morphology of the brains and by the ABC immunohistochemistry technique for the revelation of GFAP. The polyclonal antisera anti-GFAP (Dako, Glostrup, Denmark) was used at specific working dilution of 1/100 overnight at 4°C. Visualization was carried out using the Vectastain Elite ABC kit (Vector Labs, Inc., Burlingame, CA, USA) and revealed by 3 mg of 3,3’-diaminobenzidine-tetrahydrochloride (Sigma-Aldrich) in 10 mL PBS and 150 μL 3% H2O2. The sections were then counterstained with hematoxylin for 1 min. Antibody specificity was assessed by omitting the primary antisera. The images were examined and acquired by a Kontron Electronic Imaging System KS300 (Zeiss, Oberkochen, Germany).
Quantification of GFAP
An objective measure of the area contained brown pixels corresponding to immunoreactive staining for GFAP was quantified using the software ImageJ (W.S. National Institutes of Heart, Bethesda MD, USA) as described previously.29,30 Images were converted to Grey Scale “RGB stack” and green channel image used for analysis. Total area of immunostained tissue above the threshold was quantified and expressed as proportion of tissue animal total observed area. The test was replicated on serial sections of three animals for each experimental group. The data were summarized by GraphPad Prism 5.
The data of mean percentage values for the different brain areas of lizards of different experimental groups were analysed using the software GraphPad Prism 5 and the significance of differences between experimental groups was assessed by ANOVA followed by Tukey post-hoc test. Significance was taken as P<0.05.
Control lizards appeared in good health and without signs of any abnormality. None of control or treated lizards died during the course of experiment. However, in the treated lizards we observed irritability, increased aggression and a reduction in the food consumed.
The general histology of P. siculus brain in control specimens showed a homogeneous tissue with well defined structures in all the areas of the organ. In fact both the neurons and the glial cells showed a regular shape with well defined edges. The nervous tissue was well vascularized and showed a good affinity to stains (Figures 1a and 2a). The ependymocytes showed globular shape and were organized in their epithelial layer lining the ventricular lumen (Figure 1c). No particular alteration was observed in lizard brains at 2 days from treatment. Instead, after 7 and 16 days we observed a decrease of tinctorial affinity of the nervous cells in particular in the area of optic tectum, in the cerebellum (Figure 1b) and in the ependyma (Figure 1d) where the ependymocytes showed a marked cellular disorganization. A diffuse increase of oedemas (Figures 1b and 2b) was also observed in these lizards.
In the brain of P. siculus the general pattern of the GFAP-immunoreactivity was fundamentally represented by long and thick fibres, which ran from cell bodies located at the ventricular surface to the meningeal layer (Figure 3 a,b) and fibres with different course spread in brain parenchyma. Numerous GFAP-immunopositive structures were observed in the optic tectum and in the medulla oblongata (Figure 3d) with a strong staining. Thin fibres GFAP-positive were present in the telencephalon parenchyma. The type of glial cells more revealed was radial glia (Figure 3 a-d). At 2 days from injection of cadmium the pattern of GFAP immunopositive structures were generally similar to those observed in control lizards (Figure 3 e, 3h). In this group an evident decrease of immunoreactivity was revealed in the telencephalon at level of the radial fibres that are juxtaposed to meningeal surface (Figure 3f), an important structure for the homeostasis of the brain parenchyma and in the optic tectum (Figure 3g). In the latter the mean of percentage area values measured was 13.4±0.7% while after 2 days this value was significantly reduced to 9.4±1% (P<0.01) (Figure 4). In the telencephalon after two days the immunostained percentage area changed significantly from 6.76 ±1.4% of control to 3±0.1% (P<0.01) (Figure 4). In this area a strong decrease of immunoreactivity was detected up to 16 days when in some sections we did not observe positivity to anti-GFAP antibody, with a mean percentage value of immunopositive area of 1.66±1% (P<0.001) (Figures 3 and 4). At 7 and 16 days from the injection the lizard brains showed also a sensible decrease of the GFAP-immunoreaction in the ventricular areas where the mean percentage value of immunopositive area changed from 3±1% of the control to 0.56±0.4% (P<0.05) and 0.16±0.15% (P<0.05), respectively and in the optic tectum where these values were 8.3±0.6% (P<0.001) and 5.9±1.2% (P<0.001) (Figure 4). In particular this decrease was observed for the fibres running from rhombencephalic ventricle and those present in the meningeal layers of optic tectum (Figure 3 i,m). These results show that in the lizards an acute exposure to cadmium provokes morphological cellular alterations in the brain but also a decrement of the expression of GFAP marker with possible consequent damage in the function of the glial cells.
These results show the ability of Cd to damage the brain parenchyma of P. siculus. This metal was administered by a single intraperitoneal injection of CdCl2, a procedure that previously was used in the same species28 and proved to determine a progressive bioaccumulation of Cd in the brain up to 16 days from the injection,19 to induce also apoptosis of the pituitary cells,18 and suggesting a possible direct long term effect of cadmium on this organ.28 In lizard in fact this metal accumulates in kidney and ovary in the first days after a single injection, at 7 days it decreases in these organs and increases gradually in liver but also in brain.28 The oedematous alterations that we have observed are probably caused by alteration of brains blood vessels and correlate to the damages on the integrity and functions of BBB, the main cause of formation of oedemas.31 We can suppose that also in the present experimental procedure Cd was penetrated in the lizard brains by crossing the BBB and affected the organ. Cadmium is known to increase permeability of the BBB,32 penetrate and accumulate in the brain of developing and adult animals, leading to brain intracellular accumulation, cellular dysfunction, and cerebral oedema.33,34 In addition to tissue damage, Cd also increases the exposure of the brain to other potentially hazardous substances.32 The BBB is a neurovascular unit formed by endothelial cells, pericytes and astroglial endfeet.35 The blood-brain barrier of the central nervous system of the reptiles Anolis carolinensis36 and Podarcis sicula37 is based on the presence of “tight” or occluding intercellular junctions between endothelial cells similiar to mammals. The effects of Cd on endothelial cells38,39 have been already reported as well as studies performed on glial cells.40-42
Several in vivo studies have shown a range of possible effects of Cd on glial cells depending on dosage, time of exposure and experimental model used.43-45 In mammals, cadmium impairs GFAP expression in the astroglia; in fact Mori et al.43 observed a reduction of GFAP network after exposure to CdCl2 in mouse stem cell/progenitors and Kovalchuk et al.45 showed a reduction of soluble GFAP in experiments performed in rats. In this work the presence of Cd into the lizard brain parenchyma is associated to a reduction of GFAP expression, a protein that is upregulated in many cases of cerebral damage, infection and diseases.46 In literature the process of reactive astrogliosis is well known and is characterized by overexpression of GFAP, hypertrophia and proliferation of astroglia with subsequent formation of glial scar.47 Indeed, in lizard we have revealed a decrease of GFAP expression. These evidences were also previously reported in the zebrafish brain, in which histological alterations, reduction of GFAP expression and bioaccumulation were observed after Cd exposure.44,48 Several studies of astroglial cells characterization made in the last years23-27,49-52 have shown that, differently to mammals, in reptiles and fish the principal type of astroglia is radial glia. This cell type could have evidently a particular susceptibility to Cd, with a probable different reactivity to this metal compared to the astrocytes. The cause of GFAP reduction could be sought in the context of known oxidizing power of Cd.53 Furthermore, because this metal have chemical similarity to zinc and calcium it can disrupt the metabolism of such elements.54 The perturbation of these homeostases could have deleterious effects since the calcium-dependent enzymes activities have a fundamental role in regulating the stability of the GFAP.55 In the last years it was discovered that the radial glia cells at ventricular level are implicated also in the processes of adult neurogenesis and regeneration of nerve tissue in many species of vertebrates.56-58 Therefore the decrease of GFAP expression could be related to the reduction of astroglial cells functionality, consequently affecting the nervous cells homeostasis and fundamental processes for the survival of these reptiles such as adult neurogenesis and the ability to regenerate damaged tissues. These results have a considerable ecological importance considering the significant contamination of Cd at level of aquatic and terrestrial ecosystems occurred in last years as a result of massive industrial activity and poor waste management. Considering the importance of brain glial cells in life and functions of animals59 and the importance of lizards in terrestrial ecosystems this work confirms and emphasizes the importance of environmental contamination by cadmium.