Figure 1.
General morphology of the PCT.
(A–D) Nuclear staining with DAPI. (A, B) Control. (B) Higher magnification of the region within the square in A. Note the cells showing elongated nuclei (arrows) and symmetric distance between each other (insert), resembling glial cells. The arrowheads denote an unusual cell type with round nuclei. (C, D) 48 h after lesion: the damaged area has a high density of cells (bottom of the image). (D) The majority of the cells have round nuclei (D – bottom insert); note also cells with elongated nuclei in the region distal to the lesion (arrows – top insert). (E, F) Hematoxylin and eosin staining showing the morphology of hemocytes 48 h after lesion. F is a higher magnification of the square sampled in E. Note in F the hemocytes (arrows) and resident glial cells (arrowheads) within the PCT. Scale bars: A, C, E = 100 µm; B, D, F = 50 µm.
Figure 2.
Density of cells in the PCT increases after injury.
(A) Schematic illustration of both normal and injured PCT showing the cellular arrangement along the tract, including the quantified area (1 µm). The anatomical position of the tract is represented by a double-sensed arrow on the top (retina – brain). (A′) 48 h after injury, the distal part of the tract had approximately three times as many cells as the control group. Student t test revealed a significant difference between the two groups (n = 5 crabs/group; p<0.005). (B) Schematic illustration of the injured tract divided into two regions (500 µm each): proximal (P) and distal (D) to the lesion. Most cells are grouped in the damaged area 500 µm from the injury. In contrast, region D maintains the same cellular density as seen in the control. (B′) Statistical analysis shows twice as many cells in region P as in D. Student t test revealed a significant difference between the two groups (n = 5 crabs/group; p<0.05). Asterisks denote significant differences between groups.
Figure 3.
PCT attracts two different types of cells to the lesion site.
(A, B) Semithin sections of injured PCT stained with toluidine blue. (A) Region P (proximal) is surrounded by cells with typical morphology: round shape, high nucleus/cytoplasm ratio, and no granules within the cytoplasm (arrows), suggesting hyaline cells. (B) Granular hemocytes (arrows) infiltrating into the nerve fibers of the tract after eyestalk ablation – region D (distal to the lesion). Only granular cells are observed in this region intermingled with nerve fibers. (C, D) Ultrathin sections of regions P and D, respectively. (C) Hemocyte, resembling a hyaline cell, with round shape and no granules in the scarce cytoplasm. The eccentric nucleus (n) has abundant euchromatin displaying the same features as the cells shown in (A). The asterisk indicates a nerve fiber. (D) Typical granular hemocyte with numerous electron-dense granules occupying the whole cytoplasm. The nucleus (n) shows an irregularly shaped membrane and heterochromatin in the periphery. (E, F) Semigranular hemocytes infiltrated into the injured tract – region D (distal to the lesion). A small number of electron-dense granules (asterisks) occupy the whole cytoplasm; the eccentric nuclei (n) have irregular/flattened shape with abundant heterochromatin in the periphery, which is not seen in hyaline hemocytes. (G, H) Granular hemocytes are easily seen in region D and most of them show double-membrane vesicles (square) within the cytoplasm suggesting phagocytosis. This is better seen in D (higher magnification). Electron-dense granules (asterisks) with different shapes and sizes surround these vesicles. Scale bars: A, B = 10 µm; C–G = 2 µm; H = 1 µm.
Figure 4.
Circulating hemocytes and PCT cells are labeled for histamine.
(A) Histamine-positive cells are red in the hemolymph; and the nuclei, DAPI stained, are in blue. (B) Injured PCT (48 h after eyestalk ablation) showing cells labeled for histamine (red) and DAPI (blue). The majority of histamine-positive cells contain granules (arrowheads) and round nuclei. No labeling was seen in the elongated nuclei (arrow) of the cells with morphology of glial cells. Scale bars: A = 20 µm; B = 10 µm.
Figure 5.
Expression of iNOS in the PCT 48 h after lesion.
(A, B, C) Control animals. (A, D, G) DAPI stained cells; (B, E, H) iNOS immunoreacted (red); (C, F, I) Merge. No cells from control PCTs were labeled for iNOS (C – insert). (D–F) Region P (proximal) of the tract observed after DAPI nuclear staining (D), after iNOS reaction (E), and the merged image (F). The insert shows a higher magnification of the double-labeled cells. (G–I) Region D (distal) of the tract observed after DAPI staining (G), after iNOS labeling (H), and the merged image (I) revealing numerous hemocytes containing granules (arrowhead), infiltrating into the tissue. No labeling was seen in the elongated nuclei (arrows) of the cells with morphology of glial cells. The insert for I shows a higher magnification of the labeled hemocytes. Scale bars: A–I = 100 µm.
Figure 6.
iNOS and IB4 expression are colocalized in circulating hemocytes.
Hemolymph was collected from five crabs, 48(A –green) and iNOS (B – red). Only hemocytes with granules, semigranular/granular cells, showed double-labeling (insert). Scale bar: A–C = 10 µm.
Figure 7.
Lesioned area of the PCT has a high expression of iNOS, IB4, and GFAP.
(A, B, C) Controls. (A) IB4; (B) iNOS; (C) GFAP. (C) GFAP-positive cells (arrows) with round nuclei and elongated cytoplasm are seen between nerve fibers of the control PCT. (D, E) Region P (bottom of the image) showing IB4 cells (insert in E) most intensively labeled. Distal to the lesion (top of the image), IB4 labeling is weak and few cells are seen. (F, G) iNOS expression in the lesion site. Note that iNOS and IB4 expression are mainly located in the PCT edge. (H, I) Region D of the tract showing several positive glial cells labeled for GFAP. They have a different morphology from resident glial cells and are seen aligned along the tract (insert). Scale bars: A, B = 50 µm; C = 40 µm; D, E = 50 µm; F, G = 40 µm; H, I = 30 µm.
Figure 8.
Schematic diagram of the hypotheses for the source of hemocytes to the injured PCT.
Hypothesis 1: Granular/semigranular (dark blue) are the first cell type to be attracted to the injured tract. In sequence, hyaline cells (light blue) are also attracted and can differentiate in granular hemocytes as they infiltrate into the nerve fibers (due to several stimuli, such as histamine and iNOS). Hypothesis 2: Granular/semigranular hemocytes (blue) are attracted to the lesion site. Following the time course of the lesion, they infiltrate into the tract. Then, in a subacute stage, hyaline cells (red) are also attracted to the injury. In both hypotheses, we can identify two different types of cells in the PCT.