All animals were successfully treated and survived to their endpoints. No complications, related to bleeding complications, perforations or other, were noted. Vessel spasm was noted both prior to and after agent injection in all animals. Nitroglycerine was injected in the first animal to reduce spasm. However, since the vessel spasm did not resolve after administering nitro, no nitro was used in subsequent treatments. Angiography after treatment confirmed good blood flow into both renal arteries.

Follow up angiograms at 14 and 90 days from all animals showed patent renal arteries with good blood to the kidneys. At 90-days, small degree of narrowing was noted with high-dose of NW2013. All animals were surgically exposed, kidney tissue was harvested and the animals were euthanized. Renal arteries and vital organs were harvested and fixed using standard techniques.

Sub-acute histologic changes were observed in NW2013 treated renal arteries. The renal nerves appear to be selectively affected in the treated vessel without any effect on the vascular tissue in the low-dose treatment group. The nerves showed the absence of nuclei and cellular structure, and were Caspase-3 positive, indicative of neuronal apoptosis (Fig. 16.2a, b). There were a few nerves that showed focal damage and others that showed total absence of nuclei. These neuronal changes were interpreted as neurodegenerative changes following treatment with NW2013. The saline-treated (control) renal nerves failed to show any degenerative changes indicative of site-specific action of the pharmacologic agent. The neurodegenerative changes were extensive and dose dependent. The high dose treatment resulted in greater neurodegenerative changes marked by total absence of nuclei within the nerve bundles; the surrounding tissue also showed fat necrosis and some arteriolar damage. In both groups the renal arteries were normal in appearance without any significant medial, intimal or adventitial injury (Fig. 16.2c, d).

Chronic changes after local administration of NW2013 showed extensive perineural fibrosis and modest inflammation (Fig. 16.3). At low concentrations, some nerves showed vacuolar degeneration as well as mild immune cell recruitment within the nerve bundles. The Caspase-3 stains were negative at the late time points, suggesting the completion of neuronal apoptosis. The renal arteries were normal in appearance without any medial, intimal or significant adventitial injury. At high dose, the arteries had moderate to severe necrosis and degenerative changes characterized by atrophied nerve fibers with regions of more significant immune cells infiltrate including the presence of macrophages, eosinophils, lymphocytes and multinucleated giant cells.

Immunostaining against Neuro-filament protein (NFP) showed varying degrees of expression indicating structural axonal damage/loss in injured fascicles (Fig. 16.4). Immunostaining against Tyrosine hydroxylase (TH) was variable where a functional loss was detected, even in the absence of recognizable axonal change. There were no unexpected deleterious findings in the chronic phase of induced injury in the adjacent examined organs. The surrounding lymph nodes showed non-specific reactive lymphoid hyperplasia.

In summary, it may be concluded that the nerve fascicle pathology characterized by neurodegenerative histology, immune cell recruitment and perineural inflammation with fibrosis was consistent with post-injury healing in the NW2013-treated tissue. In addition, the observed differences in select neuronal markers suggest both structural injury and functional loss of nerves in bundles in a treatment-specific manner. All observed changes were restricted to the NW2013 treatment cohort and was demonstrably localized to the tissue surrounding the site of administration.

Results on kidney tissue norepinephrine (NE) levels in the renal cortex are shown in Fig. 16.5 at the 90-day time points. It is noted that local administration of neurotropic agents into the vessel wall caused a decrease in NE levels compared to controls in a dose-dependent manner.

Results from the above studies clearly demonstrate that local administration of neurotropic pharmacologic agents in particular NW2013, into renal artery walls can abrogate nerve function and induce local neurodegenerative changes in a manner that is safe to surrounding tissue. All animals survived to the designated end point, and renal arteries remained fully patent without local toxicity or deleterious effects, especially at the low dose. No significant damage was noted to the vessel wall. The neurotropic pharmacologic agents acted with specificity and selectively inducted degenerative changes exclusive to the neurons surrounding the renal artery. At 14-days after NW2013 administration, nerves in the nerve bundles lacked structure and nuclei, and showed signs of significant apoptosis, as confirmed by Caspase-3 immunostaining. At 90-days, the apoptotic process was complete and the neurons were replaced by fibrous connective tissue. Axonal degeneration was observed in some nerve bundle regions. In other bundle regions, axons remained visible but they were non-functional, as evidenced by NFP staining. Correspondingly, NW2013 treatment resulted in reduced kidney tissue-specific norepinephrine levels consistent with selective damage to neurons. NE results are equivalent to those reported in preclinical studies conducted using energy-based ablative methods.

Agent NW2013 had the optimal (most safe and effective) response at low concentrations, both at 14 and 90 days and demonstrated effective dose-response. At high concentrations, the agent was not soluble and drug particulates caused localized tissue damage characterized by necrosis. Although a higher reduction in tissue NE levels was observed at a higher dose, the lack of observed side effects in the low dose NW2013 treatment cohort favor the use of NW2013 at lower concentrations.