Sprague-Dawley rats develop progressive motor dysfunctions during the third year of life. We use this as a model to examine possible neuronal mechanism(s) that may cause motor impairments occuring during aging. In this study we have used indirect immunofluorescence histochemistry (IF) and in situ hybridization histochemistry (ISH) to study quantitatively and qualitatively the staining pattern and mRNA expression of calcitonin gene-related peptide (alpha-CGRP), growth-associated protein 43 (GAP-43), and acidic fibroblast growth factor (aFGF) in spinal lumbar motoneurons of young adult (2-3 months) and aged (30 months) Sprague-Dawley rats. In addition, mRNAs encoding choline acetyltransferase (ChAT), beta-CGRP, and cholecystokinin (CCK) were analyzed. All aged rats used in this study disclosed symptoms of hindlimb incapacity, ranging from mild weight-bearing insufficiency to paralysis of the hind limbs. The symptoms were confined to the musculature of the hindlimb and hip regions. Only a small number (approximately 15%) of the large motoneurons that innervate the hindlimb muscles were lost in those aged rats that had clinical symptoms of hindlimb motor incapacities. The remaining motoneurons expressed ChAT mRNA at levels similar to those of young adult rats. The vast majority of these motoneurons showed increased mRNA levels for alpha-CGRP and GAP-43. Aged motoneurons contained more CGRP like immunoreactivity (LI), but the number of immunoreactive neurons was smaller than in adult rats. GAP-43-LI could be detected in motoneurons in aged, but not in adult, rats. GAP-43-LI was always colocalized with CGRP-LI in aged motoneurons. Studies of individual aged rats revealed that the increase of GAP-43 mRNA-positive cell bodies occurred in cases with the most severe clinical symptoms, whereas the increase in alpha-CGRP was even evident in rats with mild symptoms. No alterations in content of aFGF-LI or aFGF mRNA could be detected in the aged rat, and the content of CCK and beta-CGRP mRNAs was also normal. The usefulness of this rat model for studies of neuromuscular aging and possible functional roles for GAP-43 and CGRP in plastic and regenerative processes during aging are discussed.

Confocal microscopes are often used to study specimens labelled with fluorophores. A commonly used method for simultaneous recording of the distribution of multiple fluorophores is to divide the fluorescent light emitted by the specimen into different wavelength regions using dichroic and bandpass filters. These different wavelength regions are then distributed to multiple detectors. However, the broad and overlapping spectra of commonly used fluorophores often result in considerable crosstalk between channels. A new technique, intensity-modulated multiplebeam scanning (IMS) microfluorometry, can be used to reduce this cross-talk substantially.The IMS technique is implemented with two laser beams of different wavelengths, intensity-modulated at different frequencies, which illuminate the specimen simultaneously. The two laser wavelengths predominantly excite one fluorophore each. Fluorescent light from the specimen is divided into two wavelength regions (red and green) which are detected by two photomultiplier tubes. The output signals from the photomultiplier tubes are connected to lock-in amplifiers. The effect of using modulated laser beams, in combination with lock-in amplifiers, is strongly to reduce cross-talk between the channels. The performance of the IMS technique using various types of specimen is compared with the results obtained using the conventional multi-detector method.

SUMMARY Studies of doubly stained specimens were performed with a confocal scanning microscope. The instrument used provides the possibility of making separate detections of the fluorescent dyes. The optimal choice of excitation wavelengths and optical filters are discussed. The fluorphores that were used are Lucifer Yellow, Texas Red, fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate.

Background It is commonly accepted that in obesity free fatty acids (FFA) cause insulin resistance and hyperglycemia, which drives hyperinsulinemia. However, hyperinsulinemia is observed in subjects with normoglycaemia and thus the paradigm above should be reevaluated. Methods We describe two studies: MD-Lipolysis, a case control study investigating the mechanisms of obesity-driven insulin resistance by a systemic metabolic analysis, measurements of adipose tissue lipolysis by microdialysis, and adipose tissue genomics; and POEM, a cohort study used for validating differences in circulating metabolites in relation to adiposity and insulin resistance observed in the MD-Lipolysis study. Findings In insulin-resistant obese with normal glycaemia from the MD-Lipolysis study, hyperinsulinemia was associated with elevated FFA. Lipolysis, assessed by glycerol release per adipose tissue mass or adipocyte surface, was similar between obese and lean individuals. Adipose tissue from obese subjects showed reduced expression of genes mediating catecholamine-driven lipolysis, lipid storage, and increased expression of genes driving hyperplastic growth. In the POEM study, FFA levels were specifically elevated in obese-overweight subjects with normal fasting glucose and high fasting levels of insulin and C-peptide. Interpretation In obese subjects with normal glycaemia elevated circulating levels of FFA at fasting are the major metabolic derangement candidate driving fasting hyperinsulinemia. Elevated FFA in obese with normal glycaemia were better explained by increased fat mass rather than by adipose tissue insulin resistance. These results support the idea that hyperinsulinemia and insulin resistance may develop as part of a homeostatic adaptive response to increased adiposity and FFA. Funding Swedish-Research-Council (2016-02660); Diabetesfonden (DIA2017-250; DIA2018-384; DIA2020-564); Novo-Nordisk-Foundation (NNF17OC0027458; NNF19OC0057174); Cancerfonden (CAN2017/472; 200840PjF); Swedish-ALF-agreement (2018-74560).

Background: There is still a lack of knowledge on long-term effects of surgical and non-surgical weight-lowering treatments. BASUN is a prospective study with 10 years of follow-up that will observe the effects and consequences of surgical and medical treatment of obesity. The aims are to cover areas where data on long-term outcomes are lacking, e.g., nutritional deficiencies, substance abuse, psychiatric health, as well as patient-reported outcomes. Methods: BASUN is a cohort study that recruited study persons with obesity (BMI ≥ 35 kg/m 2) referred to the Regional Obesity Centre of Region Västra Götaland. The interventions were Roux-en-Y gastric bypass (RYGB) or Sleeve gastrectomy (SG), or 12 months of structured, multi-professional medical treatment (MT), including very low energy diet, followed by diet and pharmaceutical treatment. The study is not randomized, but based on patients preferences and multidisciplinary assessments. The study persons are examined at baseline, 2, 5, and 10 years with blood tests, measurements and questionnaires. The recruitment period lasted from May 2015 to November 2017. Results: One thousand one hundred twenty-seven patients were included (74% female). Three hundred eighty-two patients were accepted for medical treatment, 589 for surgical treatment (388 RYGB and 201 SG) and 156 patients left the study without treatment, leaving a final study population of 971 patients. There were slight differences between the treatment groups with regards to age and BMI. Pharmaceutical treatments, level of education, smoking and marital status were not significantly different between the groups. Conclusion: This study will follow 971 obese subjects in clinical practice treated with the best surgical or medical methods currently available. It has the potential to evaluate outcomes usually not reported in short-term studies, and to assist in identifying factors that are of importance for the choices of treatment. The main limitations are non-randomization and differences in baseline characteristics. The large number of participants and the length of the prospective follow-up are major strengths of the study. BASUN is designed to identify both early and late benefits and adverse events of treatment of obesity. Trial registration: This trial was prospectively registered on March 03, 2015; NCT03152617.

The confocal scanning laser microscope (CSLM) offers improved optical resolution and contrast, high photometric precision, and the ability to make optical sections. These benefits were explored for use in quantitative analysis of immunofluorescence-labeled axon terminals. Guidelines were obtamed for adjustments ofthe CSLM parameters In the prosent applications, bleaching of the fluorescence did not represent a serious obstade to analysis with the CSLM. A method was developed to distinguish the background fluorescence from the specific fluorescence labeling. This procedure made way for the development ofautomated quantification of immunolabeled axon terminals. The automated pro-I Supported by grants from the Swedish Medical Research Council

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K E Y w O R D S. Scanning laser microscopy, confocal microscopy, fluorescence, dual labels, cross-talk, image processing, FITC, TRITC. S U M M A R YBy using dual detectors in combination with a dichroic filter, it is possible to record simultaneously the distribution of two fluorescent labels in a specimen. It is often difficult, however, to obtain a good separation, i.e. each detector will generally detect light from more than one fluorophore. In such cases it is desirable to find imageprocessing methods to improve the separation. A simple method is to form a linear combination of the recorded images. In this paper we investigate the necessary prerequisites for this method to be successful, and we also investigate to what extent these are fulfilled in some practical cases. In this context the spectral properties of the fluorophores turn out to be of crucial importance. Even when the necessary prerequisites are not strictly fulfilled, a considerable improvement in image quality can, nevertheless, be obtained.