Short-term inhalation of isoflurane improves the outcomes of intraportal hepatocyte transplantation

Abstract

Clinical hepatocyte transplantation (HTx) is only performed without general anesthesia, while inhalation anesthetics are usually used in animal experiments. We hypothesized that isoflurane may be a possible reason for the discrepancy between the results of animal experiments and the clinical outcomes of HTx. Syngeneic rat hepatocytes (1.0 × 10⁷) were transplanted to analbuminemic rats with (ISO group) and without (AW group) isoflurane. The serum albumin, AST, ALT, LDH levels and several inflammatory mediators were analyzed. Immunohistochemical staining and ex vivo imaging were also performed. The serum albumin levels of the ISO group were significantly higher in comparison to the AW group (p < 0.05). The serum AST, ALT, LDH levels of the ISO group were significantly suppressed in comparison to the AW group (p < 0.0001, respectively). The serum IL-1β, IL-10, IL-18, MCP-1, RNTES, Fractalkine and LIX levels were significantly suppressed in the ISO group. The ischemic regions of the recipient livers in the ISO group tended to be smaller than the AW group; however, the distribution of transplanted hepatocytes in the liver parenchyma was comparable between the two groups. Isoflurane may at least in part be a reason for the discrepancy between the results of animal experiments and the clinical outcomes of HTx.

Figure 1

Hepatocyte engraftment after transplantation with and without the short-term inhalation of isoflurane. The serum albumin levels of the ISO group (dotted line, n = 10) were significantly higher than those of the AW group (solid line, n = 10) (*p < 0.05, **p < 0.01). The error bars represent standard deviation.

Figure 2

The AST, ALT, and LDH levels after hepatocyte transplantation with and without the short-term inhalation of isoflurane. (A) The serum AST levels of the ISO group (dotted line, n = 9) were significantly suppressed in comparison to the AW group (solid line, n = 10) (***p < 0.0001). (B) The serum ALT levels of the ISO group (dotted line, n = 9) were significantly suppressed in comparison to the AW group (solid line, n = 10) (***p < 0.0001). (C) The serum LDH levels of the ISO group (dotted line, n = 9) were significantly suppressed in comparison to the AW group (solid line, n = 10) (***p < 0.0001). The error bars represent standard deviation.

Figure 3

Inflammatory mediator levels after hepatocyte transplantation with and without the short-term inhalation of isoflurane. The serum levels of IL-1β (A), IL-10 (B), IL-18 (C), MCP-1 (D), RANTES (E), Fractalkine (F), and LIX (G) in the ISO group (dotted line, n = 9) were significantly downregulated in comparison to the AW group (solid line, n = 10) (*p < 0.05, **p < 0.01, ***p < 0.0001). The error bars represent standard deviation.

Figure 4

Albumin staining of the transplanted hepatocytes in the AW and ISO groups. (A) A representative photomicrograph of the albumin staining. The red arrow shows an albumin-positive hepatocyte in zone 1. Yellow arrows show albumin-positive hepatocytes in zone 2. The black arrow shows an albumin-positive hepatocyte in zone 3. The white arrow shows an albumin-positive hepatocyte in the portal vein (magnification: × 100, scale bar: 200 μm, P portal vein radicles, CV central vein). Albumin-positive hepatocyte grafts were widely distributed in the liver sinusoid in both groups. (B) The total numbers of albumin-positive hepatocyte grafts at 1 day after transplantation were comparable between the AW (black bar, n = 6) and ISO (white bar, n = 6) groups. Regarding the distribution of the transplanted hepatocytes in the recipient livers, no significant differences were observed between the two groups in any area. The error bars represent standard deviation.

Figure 5

Ex vivo imaging evaluation of the transplanted hepatocytes. (A,B) A representative ex vivo image of the liver in the AW group (n = 3) at 1 day after transplantation (A: downward view of the liver, B: upward view of the liver). (C,D) A representative ex vivo image of the liver in the ISO group (n = 4) at 1 day after transplantation (C: downward view of the liver, D: upward view of the liver). The distribution pattern of the transplanted hepatocytes in the recipient livers appeared to be similar between the AW and ISO groups. The error bars represent standard deviation.

Figure 6

Evaluation of ischemic liver tissues in the AW and ISO groups. (A) A representative photomicrograph of the ischemic region (red arrow) in the liver parenchyma (magnification: × 100, scale bar: 200 μm). (B) The ischemic liver tissues tended to be more evident in the AW group (3.69 ± 1.05%, n = 6) in comparison to the ISO group (2.42 ± 1.76%, n = 6), although the difference did not reach statistical significance (p = 0.07). The error bars represent standard deviation.

Figure 7

Pictures of catheter insertion. The AW and ISO group were inserted catheter under isoflurane at a concentration of 2.0 L/min with no adjunctive analgesics. (A) Under isoflurane anesthesia, a 1 cm skin incision was made in the right lateral abdomen of the recipient rat, then a median abdominal incision was made. A subcutaneous tunnel was created to connect the median abdominal incision to the lateral abdominal incision. (B) Under isoflurane anesthesia, the catheter was inserted into the portal vein and fixed using medical Aron Alpha. The catheter was guided out of the abdomen, through the right rectus abdominis muscle and further guided to the lateral abdomen via a subcutaneous tunnel.