Stress causes increased dynorphin (DYN) expression in limbic brain regions and antagonism of kappa-opioid receptors may offer therapeutic potential for the treatment of depression. A potential site of DYN action relevant to stress and related neuropsychiatric disorders is the locus coeruleus (LC), the primary source of forebrain norepinephrine. Therefore, using immunofluorescence and immunoelectron microscopic analyses, we characterized the cellular substrates for interactions between DYN and tyrosine hydroxylase (TH), a catecholamine synthesizing enzyme in single sections through the rat LC. Light microscopic analysis of DYN immunoreactivity indicated that DYN fibers are distributed within the core and pericoerulear subregions of the LC. Using electron microscopy, immunoperoxidase labeling for DYN was primarily found in axon terminals, although in some cases was diffusely localized to somatodendritic processes. When DYN-containing axons formed synaptic contacts, they typically (89%) exhibited an asymmetric morphology. Almost a third (28%) of the postsynaptic targets of DYN-containing axons contained immunogold labeling for TH. These findings reveal some diversity as to the localization of DYN in the LC within axons that contact both TH and non-TH containing dendrites. However, the present data provide the first ultrastructural evidence that DYN-containing axon terminals directly innervate catecholaminergic LC dendrites. Moreover, DYN axon terminals targeting catecholaminergic LC dendrites via asymmetric synapses are consistent with localization within excitatory type afferents to the LC. Therefore, direct modulation of catacholaminergic LC neurons maybe an important site of action for DYN relevant to stress and stress-related disorders.

Objective Lower extremity amputation is often performed in patients where both lower extremities are at risk due to peripheral arterial disease or diabetes, yet the proportion of patients who progress to amputation of their contralateral limb is not well defined. We sought to determine the rate of subsequent amputation on both the ipsilateral and contralateral lower extremities following initial amputation. Methods We conducted a retrospective review of all patients undergoing lower extremity amputation (exclusive of trauma or tumor) at our institution from 1998 to 2010. We used International Classification of Diseases-Ninth Revision codes to identify patients and procedures as well as comorbidities. Outcomes included the proportion of patients at 1 and 5 years undergoing contralateral and ipsilateral major and minor amputation stratified by initial major vs minor amputation. Cox proportional hazards regression analysis was performed to determine predictors of major contralateral amputation. Results We identified 1715 patients. Mean age was 67.2 years, 63% were male, 77% were diabetic, and 34% underwent an initial major amputation. After major amputation, 5.7% and 11.5% have a contralateral major amputation at 1 and 5 years, respectively. After minor amputation, 3.2% and 8.4% have a contralateral major amputation at 1 and 5 years while 10.5% and 14.2% have an ipsilateral major amputation at 1 and 5 years, respectively. Cox proportional hazards regression analysis revealed end-stage renal disease (hazard ratio [HR], 3.9; 95% confidence interval [CI], 2.3–6.5), chronic renal insufficiency (HR, 2.2; 95% CI, 1.5–3.3), atherosclerosis without diabetic neuropathy (HR, 2.9; 95% CI, 1.5–5.7), atherosclerosis with diabetic neuropathy (HR, 9.1; 95% CI, 3.7–22.5), and initial major amputation (HR, 1.8; 95% CI, 1.3–2.6) were independently predictive of subsequent contralateral major amputation. Conclusions Rates of contralateral limb amputation are high and predicted by renal disease, atherosclerosis, and atherosclerosis with diabetic neuropathy. Physicians and patients should be alert to the high risk of subsequent amputation in the contralateral leg. All patients, but particularly those at increased risk, should undergo close surveillance and counseling to help prevent subsequent amputations in their contralateral lower extremity.

We previously demonstrated that the opioid peptide enkephalin and corticotropin‐releasing factor (CRF) are occasionally colocalized in individual axon terminals but more frequently converge on common dendrites in the locus coeruleus (LC). To further examine potential opioid cotransmitters in CRF afferents we investigated the distribution of pro‐opiomelanocortin (POMC), the precursor that yields the potent bioactive peptide β‐endorphin, with respect to CRF immunoreactivity using immunofluorescence and immunoelectron microscopic analyses of the LC. Coronal sections were collected through the dorsal pontine tegmentum of rat brain and processed for immunocytochemical detection of POMC and CRF or tyrosine hydroxylase (TH). POMC‐immunoreactive processes exhibited a distinct distribution within the LC as compared to the enkephalin family of opioid peptides. Specifically, POMC fibers were enriched in the ventromedial aspect of the LC with fewer fibers present dorsolaterally. Immunofluorescence microscopy showed frequent coexistence of POMC and CRF in varicose processes that overlapped TH‐containing somatodendritic processes in the LC. Ultrastructural analysis showed POMC immunoreactivity in unmyelinated axons and axon terminals. Axon terminals containing POMC were filled with numerous large dense‐core vesicles. In sections processed for POMC and TH, ∼ 29% of POMC‐containing axon terminals (n = 405) targeted dendrites that exhibited immunogold–silver labeling for TH. In contrast, sections processed for POMC and CRF showed that 27% of POMC‐labeled axon terminals (n = 657) also exhibited CRF immunoreactivity. Taken together, these data indicate that a subset of CRF afferents targeting the LC contain POMC and may be positioned to dually impact LC activity.

Background Rates of major lower extremity amputation in patients with peripheral artery disease are higher in rural communities with markers of low socioeconomic status, but most Americans live in metropolitan areas. Whether amputation rates vary within US metropolitan areas is unclear, as are characteristics of high amputation rate urban communities. Methods and Results We estimated rates of major lower extremity amputation per 100 000 Medicare beneficiaries between 2010 and 2018 at the ZIP code level among ZIP codes with ≥100 beneficiaries. We described demographic characteristics of high and low amputation ZIP codes, and the association between major amputation rate and 3 ZIP code–level markers of socioeconomic status—the proportion of patients with dual eligibility for Medicaid, median household income, and Distressed Communities Index score—for metropolitan, micropolitan, and rural ZIP code cohorts. Between 2010 and 2018, 188 995 Medicare fee‐for‐service patients living in 31 391 ZIP codes with ≥100 beneficiaries had a major lower extremity amputation. The median (interquartile range) ZIP code–level number of amputations per 100 000 beneficiaries was 262 (75–469). Though nonmetropolitan ZIP codes had higher rates of major amputation than metropolitan areas, 78.2% of patients undergoing major amputation lived in metropolitan areas. Compared with ZIP codes with lower amputation rates, top quartile amputation rate ZIP codes had a greater proportion of Black residents (4.4% versus 17.5%, P <0.001). In metropolitan areas, after adjusting for clinical comorbidities and demographics, every $10 000 lower median household income was associated with a 4.4% (95% CI, 3.9–4.8) higher amputation rate, and a 10‐point higher Distressed Communities Index score was associated with a 3.8% (95% CI, 3.4%–4.2%) higher amputation rate; there was no association between the proportion of patients eligible for Medicaid and amputation rate. These findings were comparable to the associations identified across all ZIP codes. Conclusions In metropolitan areas, where most individuals undergoing lower extremity amputation live, markers of lower socioeconomic status and Black race were associated with higher rates of major lower extremity amputation. Development of community‐based tools for peripheral artery disease diagnosis and management targeted to communities with high amputation rates in urban areas may help reduce inequities in peripheral artery disease outcomes.