Qualitative and Quantitative Study of the Changes in the Ultrastructure of Mammalian Adrenal Cortex Caused by the Venezuelan Tigra Mariposa (Bothrops venezuelensis) Snake Venom

Abstract

The damage of the adrenal gland by snake venoms needs to be clarified. Lethality (LD₅₀) of Bothrops venezuelensis (Bv) venom was established by intraperitoneally mice injections. Preparation of specimens for transmission electron microscopy samples from cortex adrenal gland biopsies at 3, 6, and 24 h was processed. The quantitative description by the principal component analysis (PCA) of the adrenal gland was as follows: thickening of the capillary endothelium, area of the capillary lumen, cell nucleus area, enlargement of the perinuclear space, number of mitochondria, area of the mitochondria, number of mitochondrial cristae, number of cristae per mitochondrial unit, and tubular diameter of the smooth endoplasmic reticulum (SER). Sections of the adrenal cortex, after 3 h postinjection with Bv venom showed in the cortical cells: mitochondria with tubular cristae and slightly swollen SER cisternae, nucleus with variable heterochromatin content, irregular edges, and swollen nuclear envelope. After 6 h, cells with swollen nucleus envelope, electron dense lipids and mitochondria with loss of their cristae were observed. Myelin figures, close to the microvilli of the cortical cell, multivesicular bodies, swollen profiles of the SER, and electron dense lipid drops were noticed. After 24 h, thickening of the endothelial wall, fenestrae and projections into the capillary lumen, loss of the mitochondrial cristae, destruction of the capillary and the plasma membrane of the cortical cell, multivesicular body, SER loss, and an enlargement of the perinuclear space were detected. In the quantitative PCA, there were significant changes after the venom treatments.

Keywords: Adrenal gland; Bothrops venezuelensis; mitochondria; principal component analysis; smooth endoplasmic reticulum; ultrastructure; venom.

Figure 1

The adrenal gland cortex normal control. The mitochondria are observed with the tubular cristae (star), central nuclei (Nu) with normal perinuclear space (arrow) associated with heterochromatin, short cisternae of the rough endoplasmic reticulum, tubular profiles of the smooth endoplasmic reticulum, Golgi (Go), lipofuscin granules in different stages of growth (triangle), capillary microvilli (oval), and a lipid drop (Lip). Micromark = 1 μm

Figure 2

Adrenal gland cortex after 3 h of Bothrops venezuelensis venom injection. (a) An erythrocyte is observed within the capillary (E), loss of the continuity of the endothelial wall (oval), projections of the capillary lumen and remains of the endothelial wall (the plasma membrane is not distinguished) (circle). The longer oval indicates a larger space between the limit of the endothelial cell and the cortical cell. In the cortical cell, the round and slightly elongated profiles correspond to the smooth endoplasmic reticulum tubular. Swollen mitochondria with scarce cristae (stars) are observed. Micromark = 1 μm. (b) In the cortical cell, a nucleus (Nu) of irregular contours with abundant heterochromatin and dilated perinuclear space (arrowhead) is noticed. There is a Golgi apparatus (Go), a clearly swollen mitochondria (star), a very swollen cisternae of rough endoplasmic reticulum (RER) and an autophagic vacuole with mitochondrial debris (triangle). Micromark = 1 μm. (c) The circular profiles of different diameters correspond to cross-sections of smooth endoplasmic reticulum. Mitochondria with tubular cristae (star) are observed. Micromark = 1 μm. (d) Lipid droplets (Lip) of different electron density are seen; swollen mitochondria (star) where a lipofucsin granule is observed adjacent to the mitochondria. Circular profiles and small tubules of smooth endoplasmic reticulum are seen). There are also profiles of larger diameter. An erythrocyte is located (E). Micromark = 1 μm

Figure 3

The adrenal gland cortex after 6 h of Bothrops venezuelensis venom injection. (a) A fibroblast with an elongated nucleus (Nu) and a swollen perinuclear space (arrow head) which lies between the plasma membrane of the cortical cell (arrow) and the capillary wall where the fenestrae are observed of it (oval). Swollen cisternae of the rough endoplasmic reticulum are observed. Above is seen the separation of the plasma membrane from the cortical cell and the fibroblast (circle). A mitochondrion (star) with tubular cristae and a lipid drop (Lip) is observed. Micromark = 1 μm. (b) A pseudoinclusion of the cytoplasm is seen, surrounded by the perinuclear space and associated to the latter by the nuclear side, heterochromatin is noticed. Micromark = 1 μm. (c) A very electron dense lipid drop (Lip) and a myelin figure (circle) are seen. The microvilli of the cortical cell (triangle) and mitochondria (star) are appreciated. The profiles of different diameter of smooth endoplasmic reticulum are observed. Micromark = 1 μm. (d) Mitochondria with swollen cristae (star) and a lipid droplet (Lip). Some profiles of different diameter of smooth endoplasmic reticulum and a multivesicular body (circle) are also seen. Micromark = 1 μm

Figure 4

The adrenal gland cortex after 24 h of Bothrops venezuelensis venom injection. (a) Inside the capillary, an erythrocyte (E) is seen. The endothelial wall shows some fenestrae (oval), and there is an area of discontinuity of the wall (circle). Several lipid drops (Lip), profiles of smooth endoplasmic reticulum as well as areas lacking smooth endoplasmic reticulum (square) are noticed. Some swollen mitochondria (star) are seen. Micromark = 1 μm. (b) The destruction of a capillary (triangle) is observed. The endothelial wall was lost (arrow). Mitochondria of the cortical cell (star) are appreciated. The multivesicular body (circle), loss of the smooth endoplasmic reticulum (square), and a lysosome (arrow head) are observed. Micromark = 1 μm. (c) The nucleus (Nu) exhibits some areas of the swollen perinuclear space (arrowhead). Micromark = 1 μm. (d) It is observed a nucleus (Nu) without heterochromatin, and a electron dense body that could be the nucleolus but with its characteristic structure lost (fibrillar centre, dense fibrillar component, and granular component) (triangle). The structure of the nuclear envelope is not distinguished, and in the cytoplasm, there is degeneration, with swollen profiles of smooth endoplasmic reticulum (circle). Mitochondria with swollen cristae (star) are observed. Micromark = 1 μm

Figure 5

Graphical representation of the principal component analyses. The points describe the temporal performance (3, 6, and 24 h) of the control group and Bothrops venezuelensis venom. The points of the coordinate system represent 95.85% of the information synthesized in the eight variables described: Area of the mitochondria (AMit), Number of mitochondria cristae (NMitC), Number of mitochondria (NMit), Area of the nucleus (NA), Diameter of cisternae of the smooth endoplasmic reticulum (DCSER), Thickening of the capillary endothelium (TCE), Capillary lumen (CL), and Thickening of perinuclear space (TPS). The autovectors (E-values) indicate the direction in which each variable (previously described) increases its magnitude

Figure 6

(a) thickening of capillary endothelium, (b) capillary lumen variations, (c) nucleus area changes, and (d) thickening of perinuclear space after 3, 6, and 24 h of Bothrops venezuelensis venom injection were evaluated by an analysis of variance of two factors, which was developed at 95% confidence. It was found that there were stastistically significant differences for changes in the treatments (factor 1) P = 0. 00 and significant difference for times (factor 2) P = 0.00, with an interaction of P = 0.00. The figure shows the tendencies of the means by the two treatments over time. The values are showed in Table 1

Figure 7

(a) number of mitochondria fluctuations, (b) mitochondria area variations, (c) number of mitochondria cristae, and (d) diameter of the smooth endoplasmic reticulum cisternae after 3, 6, and 24 h of Bothrops venezuelensis venom injection were evaluated by an analysis of variance of two factors at 95% confidence. It was found that there were statistically significant differences for changes in treatments (factor 1) P = 0.00 and significant difference for times (factor 2) P = 0.00, with an interaction of P = 0.00. The figure shows the tendencies of the means by the two treatments over time. The values are showed in Table 1