Ivica Grković

A Comprehensive Evaluation of Potential Lung Function Associated Genes in the SpiroMeta General Population Sample

Rationale Lung function measures are heritable traits that predict population morbidity and mortality and are essential for the diagnosis of chronic obstructive pulmonary disease (COPD). Variations in many genes have been reported to affect these traits, but attempts at replication have provided conflicting results. Recently, we undertook a meta-analysis of Genome Wide Association Study (GWAS) results for lung function measures in 20,288 individuals from the general population (the SpiroMeta consortium). Objectives To comprehensively analyse previously reported genetic associations with lung function measures, and to investigate whether single nucleotide polymorphisms (SNPs) in these genomic regions are associated with lung function in a large population sample. Methods We analysed association for SNPs tagging 130 genes and 48 intergenic regions (+/−10 kb), after conducting a systematic review of the literature in the PubMed database for genetic association studies reporting lung function associations. Results The analysis included 16,936 genotyped and imputed SNPs. No loci showed overall significant association for FEV1 or FEV1/FVC traits using a carefully defined significance threshold of 1.3×10−5. The most significant loci associated with FEV1 include SNPs tagging MACROD2 (P = 6.81×10−5), CNTN5 (P = 4.37×10−4), and TRPV4 (P = 1.58×10−3). Among ever-smokers, SERPINA1 showed the most significant association with FEV1 (P = 8.41×10−5), followed by PDE4D (P = 1.22×10−4). The strongest association with FEV1/FVC ratio was observed with ABCC1 (P = 4.38×10−4), and ESR1 (P = 5.42×10−4) among ever-smokers. Conclusions Polymorphisms spanning previously associated lung function genes did not show strong evidence for association with lung function measures in the SpiroMeta consortium population. Common SERPINA1 polymorphisms may affect FEV1 among smokers in the general population.

Effects of Tensioning the Lumbar Fasciae on Segmental Stiffness During Flexion and Extension : Young Investigator Award Winner

Study Design. Biomechanical study of unembalmed human lumbar segments. Objective. To investigate the effects of tensioning the lumbar fasciae (transversus abdominis [TrA]) aponeurosis) on segment stiffness during flexion and extension. Summary of Background Data. Animal and human studies suggest that TrA may influence intersegmental movement via tension in the middle and posterior layers of lumbar fasciae (MLF, PLF). Methods. Compressive flexion and extension moments were applied to 17 lumbar segments from 9 unembalmed cadavers with 20 N lateral tension of the TrA aponeurosis during: 1) “static” tests: load was compared when fascial tension was applied during static compressive loads into flexion-extension; 2) “cyclic loading” tests: load, axial displacement, and stiffness were compared during repeated compressive loading cycles into flexion-extension. After testing, the PLF was incised to determine the tension transmitted by each layer. Results. At all segments and loads (<200 N), fascial tension increased resistance to flexion loads by ∼9.5 N. In 15 of 17, fascial tension decreased resistance to extension by ∼6.6 N. Fascial tension during cyclic flexion loading decreased axial displacement by 26% at the onset of loading (0–2 N) and 2% at 450 N (13 of 17). During extension loading, fascial tension increased displacement at the onset of loading (10 of 17) by ∼23% and slightly (1%) decreased displacement at 450 N. Segment stiffness was increased by 6 N/mm in flexion (44% at 25 N) and decreased by 2 N/mm (8% at 25 N) in extension. More than 85% of tension was transmitted through the MLF. Conclusions. Tension on the lumbar fasciae simulating moderate contraction of TrA affects segmental stiffness, particularly toward the neutral zone.

CALBINDIN D28K-IMMUNOREACTIVITY IDENTIFIES DISTINCT SUBPOPULATIONS OF SYMPATHETIC PRE- AND POSTGANGLIONIC NEURONS IN THE RAT

Neurons performing the same function can be identified immunohistochemically because they often share the same neurochemistry. The distribution of calcium‐binding proteins, like calbindin, has been used previously to identify functional subpopulations of neurons in many parts of the nervous system. In this study we have investigated the distribution of calbindin D28K‐immunoreactivity in subpopulations of sympathetic preganglionic neurons in the intermediolateral nucleus of the rat spinal cord. The majority of calbindin D28K‐immunoreactive preganglionic neurons also had co‐localised nitric oxide synthase, although a population of preganglionic neurons in the mid‐ to low thoracic intermediolateral nucleus expressed only calbindin D28K‐immunoreactivity. Retrograde‐tracing studies showed that calbindin D28K‐immunoreactive neurons projected to the superior cervical and stellate ganglia, with smaller numbers of cells projecting to the lumbar sympathetic chain and superior mesenteric ganglia. Very few calbindin D28K‐immunoreactive neurons projected to the inferior mesenteric ganglion, and none projected to the adrenal medulla. The distribution of calbindin D28K‐immunoreactive terminals and postganglionic neurons in the superior cervical and stellate ganglia was also investigated. Many postganglionic neurons were calbindin D28K‐immunoreactive, and most of these lacked neuropeptide Y‐immunoreactivity. Calbindin D28K‐immunoreactive nerve terminals were common and formed dense pericellular baskets around many postganglionic neurons, including some of those that were calbindin D28K‐immunoreactive, but only rarely formed pericellular baskets around neuropeptide Y‐immunoreactive neurons. The function of some of the classes of postganglionic neurons that were the target of calbindin D28K‐immunoreactive preganglionic terminals was determined by combining immunohistochemistry with retrograde‐tracer injections into a range of peripheral tissues. Calbindin D28K‐immunoreactive nerve terminals, with co‐localised nitric oxide synthase‐immunoreactivity, surrounded secretomotor neurons projecting to the submandibular salivary gland and pilomotor neurons projecting to skin, but did not surround neurons projecting to brown fat or vasomotor neurons projecting to the skin, muscle, or salivary glands. J. Comp. Neurol. 386:245–259, 1997.

Immunohistochemical analysis of intracardiac ganglia of the rat heart

The neurochemistry of intracardiac neurons in whole-mount preparations of the intrinsic ganglia was investigated. This technique allowed the study of the morphology of the ganglionated nerve plexus found within the atria as well as of individual neurons. Intracardiac ganglia formed a ring-like plexus around the entry of the pulmonary veins and were interconnected by a series of fine nerve fibres. All intracardiac neurons contained immunoreactivity to PGP-9.5, choline acetyl transferase (ChAT) and neuropeptide Y (NPY). Two smaller subpopulations were immunoreactive to calbindin or nitric oxide synthase. Furthermore, a subpopulation (approximately 6%) of PGP-9.5/ChAT/NPY-immunoreactive cells lacking both calbindin and nitric oxide synthase (NOS) was surrounded by pericellular baskets immunoreactive to ChAT and calbindin. Vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activated peptide (PACAP), substance P and tyrosine hydroxylase (TH) immunoreactivity was observed in nerve fibres within the ganglion, but never in neuronal somata. Furthermore, immunoreactivity for NPY was not observed in pericellular baskets surrounding intracardiac neurons, despite being present in all intrinsic neuronal cell bodies. Taken together, the results of this study indicate a moderate level of chemical diversity within the intracardiac neurons of the rat. Such chemical diversity may reflect functional specialisation of neurons in the intracardiac ganglia.

Calretinin-containing preganglionic nerve terminals in the rat superior cervical ganglion surround neurons projecting to the submandibular salivary gland

The distribution and targets of calretinin-immunoreactive preganglionic nerve terminals in the superior cervical ganglion of the rat were examined using immunohistochemistry and retrograde neuronal tracing. Calretinin-immunoreactive nerve terminals were found throughout the ganglion, forming distinct pericellular baskets around a sub-population of postganglionic neurons. The targets of postganglionic neurons surrounded by calretinin-immunoreactive nerve terminals were determined after injection of tracer into the submandibular salivary gland, the extra-orbital lacrimal gland, the thyroid gland, the anterior chamber of the eye or the skin of the forehead. Only when tracer was injected into the submandibular gland were neurons labelled that were surrounded by calretinin-immunoreactive nerve terminals. When immunohistochemistry using antisera to neuropeptide Y was combined with retrograde tracing, only submandibular gland projecting neurons lacking neuropeptide Y were surrounded by calretinin-immunoreactive terminals. When retrograde neuronal tracer was injected into the superior cervical ganglion, a proportion of retrogradely-labelled neurons in the upper thoracic spinal cord showed relatively weak calretinin-immunoreactivity. All calretinin-immunoreactive terminals in the superior cervical ganglion disappeared following section of the sympathetic chain distal to the superior cervical ganglion. Thus, calretinin is present in a population of preganglionic neurons projecting exclusively to neuropeptide Y non-immunoreactive (presumably secretomotor) neurons innervating the submandibular salivary gland of the rat.

Relationship of NK3 receptor-immunoreactivity to subpopulations of neurons in rat spinal cord.

The distribution of immunoreactivity to the neurokinin3 receptor (NK3R) was examined in segments C7, T11‐12, L1‐2, and L4‐6 of the rat spinal cord. NK3R immunoreactivity was visualized by using two antisera generated against sequences of amino acids contained in the C‐terminal region of the NK3R. NK3R‐immunoreactive cells were numerous in the substantia gelatinosa of all spinal segments examined as well as the dorsal commissural nucleus of spinal segments L1‐2. Isolated, immunoreactive cells were scattered throughout other regions of the spinal cord. The relationship of NK3R‐immunoreactivity with neurons was demonstrated by colocalization with microtubule associated protein 2‐immunoreactivity in individual cells. Within neurons, NK3R‐immunoreactivity was associated predominately with the plasma membrane of cell bodies and dendrites. Within the substantia gelatinosa, 86% of nitric oxide synthase (NOS)‐immunoreactive neurons were also NK3R‐immunoreactive. Although NOS‐immunoreactive neurons were found throughout all other regions of the spinal cord in the segments examined, these were not NK3R‐immunoreactive. When preganglionic sympathetic neurons in spinal segments T11‐12 and L1‐2 were visualized by intraperitoneal injection of Fluorogold, less than 1% of the Fluorogold‐labeled neurons were also immunoreactive for NK3R. The large number of NK3R‐immunoreactive neurons in the substantia gelatinosa suggests that some effects of tachykinins on somatosensation may be mediated by NK3R. J. Comp. Neurol. 381:439‐448, 1997.

Separate neurochemical classes of sympathetic postganglionic neurons project to the left ventricle of the rat heart.

The sympathetic innervation of the rat heart was investigated by retrograde neuronal tracing and multiple label immunohistochemistry. Injections of Fast Blue made into the left ventricular wall labelled sympathetic neurons that were located along the medial border of both the left and right stellate ganglia. Cardiac projecting sympathetic postganglionic neurons could be grouped into one of four neurochemical populations, characterised by their content of calbindin and/or neuropeptide Y (NPY). The subpopulations of neurons contained immunoreactivity to both calbindin and NPY, immunoreactivity to calbindin only, immunoreactivity to NPY only and no immunoreactivity to calbindin or NPY. Sympathetic postganglionic neurons were also labelled in vitro with rhodamine dextran applied to the cut end of a cardiac nerve. The same neurochemical subpopulations of sympathetic neurons were identified by using this technique but in different proportions to those labelled from the left ventricle. Preganglionic terminals that were immunoreactive for another calcium-binding protein, calretinin, preferentially surrounded retrogradely labelled neurons that were immunoreactive for both calbindin and NPY. The separate sympathetic pathways projecting to the rat heart may control different cardiac functions.

Chemically distinct preganglionic inputs to iris-projecting postganglionic neurons in the rat: A light and electron microscopic study

Individual autonomic postganglionic neurons are surrounded by pericellular baskets of preganglionic terminals that are easily identifiable with the light microscope. It has been assumed that the target cell of a pericellular basket of preganglionic terminals is the neuron at the centre of the basket. This assumption has enabled the connectivity of preganglionic neurons to be determined at the light microscopic level. However, if the preganglionic terminals in a pericellular basket make synapses with the dendrites of nearby, but functionally different, postganglionic neurons, then the conclusions of light microscopic studies are far less certain. We have used a serial section ultrastructural study to determine the target of the preganglionic pericellular basket in a situation where the apparent target cell is surrounded by neurons of dissimilar function. In the rat superior cervical ganglion, postganglionic neurons projecting to the iris were identified, using retrograde tracers, as single neurons (i.e., not in clusters). We have used immunohistochemistry to show that iris‐projecting neurons are surrounded by preganglionic nerve terminals containing calcitonin gene‐related peptide (CGRP). We have demonstrated that the pericellular basket of CGRP‐immunoreactive preganglionic terminals provides inputs only to the soma at the centre of the basket and not to the dendrites of surrounding neurons. This suggests that, in autonomic ganglia, light microscopic identification of the preganglionic terminal baskets is likely to be a reliable method for identifying the targets of subclasses of preganglionic neurons. J. Comp. Neurol. 412:606–616, 1999.

Misidentification of cardiac vagal pre-ganglionic neurons after injections of retrograde tracer into the pericardial space in the rat

We evaluated whether pericardial injections of the retrograde tracers cholera toxin subunit B (CTb) or Fast Blue (FB) reliably labelled cardiac vagal pre-ganglionic neurons. Injections of CTb into the pericardial space of the rat labelled neurons in both the external and compact formations of the nucleus ambiguus. Most labelled neurons were found in the compact formation of the nucleus ambiguus, and the majority of these, and only these, expressed immunoreactivity for calcitonin gene-related peptide. This distribution of labelled neurons and their immunohistochemical properties is characteristic of oesophageal motoneurons. Examination of the oesophagus following intra-pericardial CTb applications revealed strong labelling of motor end plates within the skeletal muscle of the thoracic but not the abdominal oesophagus. When a second retrograde tracer, FB, was injected into the abdominal oesophagus, labelled somata were found adjacent to CTb-labelled neurons in the compact formation of the nucleus ambiguus. No co-localisation of tracers was found, but identical proportions of calcitonin gene-related peptide (CGRP) immunoreactivity were observed in both groups of neurons. FB injected into the pericardial space labelled intra-cardiac neurons but not brainstem neurons. We conclude that intra-pericardial, and perhaps sub-epicardial, injections of some retrograde tracers are likely to label a subset of oesophageal, as well as cardiac, vagal motor neurons in the brainstem.

Characterization of spinal afferent neurons projecting to different chambers of the rat heart

The pattern of distribution of spinal afferent neurons (among dorsal root ganglia-DRGs) that project to anatomically and functionally different chambers of the rat heart, as well as their morphological and neurochemical characteristics were investigated. Retrograde tracing using a patch loaded with Fast blue (FB) was applied to all four chambers of the rat heart and labeled cardiac spinal afferents were characterized by using three neurochemical markers. The majority of cardiac projecting neurons were found from T1 to T4 DRGs, whereas the peak was at T2 DRG. There was no difference in the total number of FB-labeled neurons located in ipsilateral and contralateral DRGs regardless of the chambers marked with the patch. However, significantly more FB-labeled neurons projected to the ventricles compared to the atria (859 vs. 715). The proportion of isolectin B(4) binding in FB-labeled neurons was equal among all neurons projecting to different heart chambers (2.4%). Neurofilament 200 positivity was found in greater proportions in DRG neurons projecting to the left side of the heart, whereas calretinin-immunoreactivity was mostly represented in neurons projecting to the left atrium. Spinal afferent neurons projecting to different chambers of the rat heart exhibit a variety of neurochemical phenotypes depending on binding capacity for isolectin B(4) and immunoreactivity for neurofilament 200 and calretinin, and thus represent important baseline data for future studies.