Figure 8.

Evidence of neuronal connections to the splenic mesothelium.

Panel a; Thin connections to the splenic capsule can be observed in trichrome stained rat spleens at low power (original magnification 5×) that are not observed by the naked eye (arrow). The origin of these connections is uncertain but they appear to be common at the splenic poles and along the inferior edge of the spleen surface.

Panel b; A connection at higher magnification (original magnification 40×). Trichrome stained tissue connects on the capsular surface often forming a ‘raised nodule’ which protrudes from the capsule more than the surrounding mesothelial layer.

Panel c; These connections stain strongly positive for the pan neuronal marker PGP9.5. Shown is a connection from a control rat stained for PGP9.5. Note also positive staining on the surrounding mesothelial layer. This was present only on the inferior axis of the spleen and was most evident in areas where connections such as this were observed. Original magnification 40×.

Panel d; PGP9.5 staining of capsular mesothelial cells in a surgical sham rat. Note the flattened appearance of the mesothelial cells and strong positive staining for PGP9.5. Original image magnification 40×.

Panel e; These connections stain negative positive for the sympathetic neuronal marker tyrosine hydroxylase. Note strong positive staining was observed around blood vessels in the splenic parenchyma (one can be observed directly below the intersection point of this connection). Original image magnification 20×.

Panel f; Acetylcholine esterase staining within a connection to the splenic capsule in a control rat. Note punctate positive staining for acetylcholine esterase throughout the connection. Positive staining for acetylcholine esterase can also be observed in the surrounding capsular mesothelial cells and underlying splenic parenchyma. Original image magnification 40×.

Panel g; PGP9.5 staining at the inferior edge of the spleen. Mesothelial cells in this region stain strongly positive for the pan neuronal marker PGP9.5. Original image magnification 5×.

Panel h; Tyrosine hydroxylase staining at the inferior edge of the spleen. Mesothelial cells in this region stain negative for the sympathetic neuronal marker tyrosine hydroxylase. Original image magnification 5×.

Panel I; Acetylcholine esterase staining at the inferior edge of the spleen. Mesothelial cells in this region stain strongly positive for the para-sympathetic neuronal marker acetylcholine esterase. Original image magnification 5×.