Double Labelling Immunohistochemistry on the Paravertebral Ganglion Related to the Smooth Vasal Musculature of the Swine Cremaster Muscle

Abstract

Retrograde neuronal tracing and double labelling immunohistochemistry methods were used to study the co‐existence of tyrosine hydroxylase (TH) with vesicular acetylcholine transporter (VAChT), neuronal nitric oxyde synthase (nNOS), calcitonine gene related peptide (CGRP), leucine‐enkephaline (LENK), neuropeptide Y (NPY), substance P (SP) and vasoactive intestinal polypeptide (VIP) in the peripheral paravertebral neurons projecting to the smooth vasal musculature of the swine cremaster muscle (CM). The fluorescent tracer Fast Blue (FB) was injected into the left CM of 4 50 Kg non‐emasculated pigs. After 7‐day survival time, the ipsilateral paravertebral L4 (PaGL4) ganglion, the most positive among the autonomic ganglia, was collected. 12 μm‐thick cryostat sections were observed under a fluorescent microscope. In the PaGL4, there were 575.5 ± 152.93 FB+ neurons. On 206 ± 51.07 FB+ cells tested for TH/ VAChT, 24.52% ± 5.6% were TH+/VAChT+, 33.65% ± 7.33% TH+/VAChT‐, 7.46% ± 2.77% TH‐/VAChT+, 34.38 ± 2.66% TH‐/VAChT‐. 211.5 ± 47 FB+ neurons were tested for TH/nNOS: 24.72% ± 5.43% were TH+/nNOS+; 29.35% ± 7.71% were TH+/nNOS‐; 7.23% ± 3.22% were TH‐/nNOS+; 38.7% ± 2.88% were TH‐/nNOS‐. On 213.25 ± 71.82 positive neurons tested for TH/CGRP, 29.4% ± 4.36% were TH+/CGRP+, 29.09% ± 7.61% TH+/CGRP‐, 11.45% ± 4.43% TH‐/CGRP+, 30.06% ± 3.7% TH‐/CGRP‐. On 248.5 ± 73.82 FB+ neurons tested for TH/LENK, 37% ± 4.25% were TH+/LENK+, 40.52% ± 5.96% TH+/LENK‐, 3.54%± 1.37% TH‐/LENK+, 18.94% ± 2.09% TH‐/LENK‐. 210 ± 53.22 FB+ cells were tested for TH/NPY: 50.72 ± 2.8% were TH+/NPY+; 15.38% ± 2.55% TH+/NPY‐; 11.12%±4.32 TH‐/NPY+; 22.79 ± 2.78% TH‐/NPY‐. 218.75 ± 65.02 of positive cells were tested for TH/SP: 26.76 ± 4.73 were TH+/SP+; 36.82% ± 7.58 TH+/SP‐; 3.78% ± 2.43% TH‐/SP+; 32.64 ± 3.75% TH‐/SP‐. On 202.5 ± 47.65 FB+ neurons tested for TH/VIP, 30.95% ± 8.2% were TH+/VIP+, 29.51% ± 3.87 TH+/VIP‐, 8.38%± 2.33% TH‐/VIP+, 31.16%± 6.04% TH‐/VIP‐. The obtained results allow us to document that the neurochemical content of the paravertebral neurons projecting to the CM differ considerably. The contemporaneous presence of adrenergic, peptidergic, nitrergic, and cholinergic neurons shows the complex role that these autonomic ganglia have in the control of smooth vasal musculature.