Age-related changes of neurochemically different subpopulations of cardiac spinal afferent neurons in rats

This study investigated the effect of aging on cardiac spinal afferent neurons in the rat. A patch loaded with retrograde tracer Fast Blue (FB) was applied to all chambers of the rat heart. Morphological and neurochemical characteristics of labeled cardiac spinal afferent neurons were assessed in young (2 months) and old (2 years) rats using markers for likely unmyelinated (isolectin B4; IB4) and myelinated (neurofilament 200; N52) neurons.     

刺激外周神经对幼鼠脊髓、海马c-fos蛋白表达的影响

目的 探讨电刺激坐骨神经后不同时间幼鼠脊髓、海马神经元c-fos蛋白的表达及意义.方法 将30只SD雄性幼鼠随机分成3组:空白对照组,单个电刺激组,强直电刺激组.强直电刺激组包括:强直刺激后1h组、2h组和3h组.首先对单个电刺激组施加单个电刺激,对强直刺激组施加强直电刺激,而后再对强直电刺激组施加同样参数的单个电刺激.实验完毕后取各组动物脊髓和海马神经元做免疫组织化学染色,观察各组动物脊髓和海马神经元内c-fos表达的变化.结果 单个电刺激组、强直电刺激组海马、脊髓神经元内c-fos蛋白强表达,与空白对照组的弱表达比较差异均有统计学意义(P＜0.05);强直刺激后,海马、脊髓不同时间神经元内的c-fos表达不同,以强直刺激后2h为表达量最高.结论 电刺激坐骨神经可使幼鼠海马、脊髓神经元内c-fos蛋白表达增加,推测电刺激后海马、脊髓神经元内c-fos表达量的增加与学习记忆联系密切.     

A subset of spinal dorsal horn interneurons crucial for gating touch-evoked pain-like behavior

A cardinal, intractable symptom of neuropathic pain is mechanical allodynia, pain caused by innocuous stimuli via low-threshold mechanoreceptors such as Aβ fibers. However, the mechanism by which Aβ fiber-derived signals are converted to pain remains incompletely understood. Here we identify a subset of inhibitory interneurons in the spinal dorsal horn (SDH) operated by adeno-associated viral vectors incorporating a neuropeptide Y promoter (AAV-NpyP⁺) and show that specific ablation or silencing of AAV-NpyP⁺ SDH interneurons converted touch-sensing Aβ fiber-derived signals to morphine-resistant pain-like behavioral responses. AAV-NpyP⁺ neurons received excitatory inputs from Aβ fibers and transmitted inhibitory GABA signals to lamina I neurons projecting to the brain. In a model of neuropathic pain developed by peripheral nerve injury, AAV-NpyP⁺ neurons exhibited deeper resting membrane potentials, and their excitation by Aβ fibers was impaired. Conversely, chemogenetic activation of AAV-NpyP⁺ neurons in nerve-injured rats reversed Aβ fiber-derived neuropathic pain-like behavior that was shown to be morphine-resistant and reduced pathological neuronal activation of superficial SDH including lamina I. These findings suggest that identified inhibitory SDH interneurons that act as a critical brake on conversion of touch-sensing Aβ fiber signals into pain-like behavioral responses. Thus, enhancing activity of these neurons may offer a novel strategy for treating neuropathic allodynia.

Damage to the nervous system by cancer, diabetes, chemotherapy, infection, or traumatic injury causes neuropathic pain, a highly debilitating chronic pain condition (1). A cardinal symptom of neuropathic pain is mechanical allodynia, pain that is produced by innocuous mechanical stimulus, such as light touch. The mechanisms underlying mechanical allodynia are poorly understood. Currently available treatments including opioids are largely ineffective.Light mechanical information from the skin is conveyed to the spinal dorsal horn (SDH) via primary afferent low-threshold mechanoreceptors (LTMRs), such as Aβ fibers. These LTMRs are considered to mediate mechanical allodynia (2–7). A major question is where and how touch signals are pathologically converted to pain in the context of nerve damage. One potential region could be the SDH where Aβ fibers and nociceptors interact through interneurons (6, 8–10), as depicted in the gate control theory of pain (11). Over the last 5 y, studies using multiple lines of transgenic mice have identified several subsets of excitatory and inhibitory interneurons in the SDH that are genetically defined (12–18) and shown that these subsets are involved in peripheral nerve injury (PNI)-induced mechanical hypersensitivity (assessed using von Frey filaments). However, the behavioral hypersensitivity by these filaments involves activation not only of LTMRs but also of nociceptors (19–24) and is effectively suppressed by treatment with morphine (18, 25). Thus, the mechanisms underlying LTMR-derived and morphine-resistant neuropathic allodynia are still poorly understood.Using a transgenic rat line W-TChR2V4 in which channelrhodopsin-2 (ChR2) was expressed at nerve endings associated with Merkel cells and lamellar cells to form Meissner’s corpuscle-like structures in the skin (26), we recently reported that following PNI, stimulation of touch-sensing Aβ fibers by illuminating the rats with blue light elicited morphine-resistant mechanical allodynia-like responses (27). Furthermore, Aβ fiber stimulation after PNI causes activation of lamina I neurons, which are normally silent in response to this stimulation. This raises the possibility that alterations in SDH circuits after PNI underscore the conversion of Aβ fiber-derived signals to morphine-resistant pain, but the underlying mechanisms remain to be determined. Considering previous findings (6, 8–11), a possible mechanism for the conversion might involve a loss or reduction of the activity of inhibitory interneurons in the SDH. A single-cell RNA sequencing study has shown that SDH inhibitory interneurons are genetically divided into over 10 subsets, some of which express mRNA encoding neuropeptide Y (NPY) (28). In immunohistochemistry, NPY has been shown to be expressed in inhibitory interneurons (29, 30). Furthermore, previous studies have demonstrated that spinal NPY has inhibitory effects on chronic pain, including PNI-induced mechanical hypersensitivity (31, 32). Thus, to identify a subset of inhibitory SDH interneurons that contributes to the behavioral symptom evoked by optical stimulation of the primary afferent Aβ fibers in the W-TChR2V4 rats after PNI, we focused on the role of inhibitory SDH interneurons operated by an adeno-associated viral (AAV) vector including a Npy promoter (AAV-NpyP). Using the optogenetic approach for neuropathic allodynia (27) combined with chemogenetics, electrophysiology, and transsynaptic tracing, this study reveals that diminished inhibitory tone of these SDH neurons operated by AAV-NpyP contributes to Aβ fiber-derived neuropathic pain-like behavioral responses. Our findings suggest that this subset of neurons could be a therapeutic target for treating neuropathic mechanical allodynia.     

Loss of Hoxb8 alters spinal dorsal laminae and sensory responses in mice

Although Hox gene expression has been linked to motoneuron identity, a role of these genes in development of the spinal sensory system remained undocumented. Hoxb genes are expressed at high levels in the dorsal horn of the spinal cord. Hoxb8 null mutants manifest a striking phenotype of excessive grooming and hairless lesions on the lower back. Applying local anesthesia underneath the hairless skin suppressed excessive grooming, indicating that this behavior depends on peripheral nerve activity. Functional ablation of mouse Hoxb8 also leads to attenuated response to nociceptive and thermal stimuli. Although spinal ganglia were normal, a lower postmitotic neural count was found in the dorsalmost laminae at lumbar levels around birth, leading to a smaller dorsal horn and a correspondingly narrowed projection field of nociceptive and thermoceptive afferents. The distribution of the dorsal neuronal cell types that we assayed, including neurons expressing the itch-specific gastrin-releasing peptide receptor, was disorganized in the lumbar region of the mutant. BrdU labeling experiments and gene-expression studies at stages around the birth of these neurons suggest that loss of Hoxb8 starts impairing development of the upper laminae of the lumbar spinal cord at approximately embryonic day (E)15.5. Because none of the neuronal markers used was unexpressed in the adult dorsal horn, absence of Hoxb8 does not impair neuronal differentiation. The data therefore suggest that a lower number of neurons in the upper spinal laminae and neuronal disorganization in the dorsal horn underlie the sensory defects including the excessive grooming of the Hoxb8 mutant.     

Neuronal calcium-binding proteins 1/2 localize to dorsal root ganglia and excitatory spinal neurons and are regulated by nerve injury

Neuronal calcium (Ca²⁺)-binding proteins 1 and 2 (NECAB1/2) are members of the phylogenetically conserved EF-hand Ca²⁺-binding protein superfamily. To date, NECABs have been explored only to a limited extent and, so far, not at all at the spinal level. Here, we describe the distribution, phenotype, and nerve injury-induced regulation of NECAB1/NECAB2 in mouse dorsal root ganglia (DRGs) and spinal cord. In DRGs, NECAB1/2 are expressed in around 70% of mainly small- and medium-sized neurons. Many colocalize with calcitonin gene-related peptide and isolectin B4, and thus represent nociceptors. NECAB1/2 neurons are much more abundant in DRGs than the Ca²⁺-binding proteins (parvalbumin, calbindin, calretinin, and secretagogin) studied to date. In the spinal cord, the NECAB1/2 distribution is mainly complementary. NECAB1 labels interneurons and a plexus of processes in superficial layers of the dorsal horn, commissural neurons in the intermediate area, and motor neurons in the ventral horn. Using CLARITY, a novel, bilaterally connected neuronal system with dendrites that embrace the dorsal columns like palisades is observed. NECAB2 is present in cell bodies and presynaptic boutons across the spinal cord. In the dorsal horn, most NECAB1/2 neurons are glutamatergic. Both NECAB1/2 are transported into dorsal roots and peripheral nerves. Peripheral nerve injury reduces NECAB2, but not NECAB1, expression in DRG neurons. Our study identifies NECAB1/2 as abundant Ca²⁺-binding proteins in pain-related DRG neurons and a variety of spinal systems, providing molecular markers for known and unknown neuron populations of mechanosensory and pain circuits in the spinal cord.Calcium (Ca²⁺) plays a crucial role in many and diverse cellular processes, including neurotransmission (1). Glutamate and neuropeptides are neurotransmitters released from the central terminals of dorsal root ganglion (DRG) neurons in the spinal dorsal horn, where signals for different sensory modalities, including pain, are conveyed to higher centers (2–12). Neurotransmitter release is tightly regulated by Ca²⁺-dependent SNARE proteins whose activity is regulated by Ca²⁺-binding proteins (CaBPs) (1, 7, 13).Parvalbumin (PV), calbindin D-28K (CB), calretinin (CR), and secretagogin (Scgn) are extensively studied EF-hand CaBPs, and they have also emerged as valuable anatomical markers for morphologically and functionally distinct neuronal subpopulations (14–17). The expression of CaBPs in DRG neurons has been thoroughly studied (18). Moreover, neuronal Ca²⁺ sensor 1 and downstream regulatory element-antagonist modulator (DREAM) are also EF-hand Ca²⁺-binding proteins in DRGs and the spinal cord (19, 20). Despite these advances, a CaBP has so far not been characterized in the majority of small- and medium-sized DRG neurons, many of which represent nociceptors.The subfamily of neuronal Ca²⁺-binding proteins (NECABs) consists of three members (NECAB1–NECAB3), probably as a result of gene duplication (21). NECABs are also EF-hand proteins, with one pair of EF-hand motifs in the N terminus and a putative antibiotic biosynthesis monooxygenase domain in the C terminus, which are linked by a NECAB homogeneous region (22). NECAB1/2 are restricted to the nervous system, whereas NECAB3 is also expressed in the heart and skeletal muscle (21).NECAB1 was first identified as the target protein of synaptotagmin I C2A-domain by affinity chromatography, with its expression restricted to layer 4 cortical pyramidal neurons, inhibitory interneurons, and hippocampal CA2 pyramidal cells in mouse brain (21, 23). The gene of the second member was cloned from mouse and initially named Necab. It encodes a 389-aa (NECAB2) (24). NECAB2 was identified as a downstream target of Pax6 in mouse retina, which is involved in retinal development (24, 25), as well as being a binding partner for the adenosine A_2A receptor (22). Furthermore, an interaction between NECAB2 and metabotropic glutamate receptor 5 (mGluR5) was demonstrated in rat hippocampal pyramidal cells, possibly regulating mGluR5’s coupling to its signaling machinery (26). Finally, NECAB3, also known as XB51, was isolated as an interacting target for the neuron-specific X11-like protein and is possibly involved in the pathogenesis of Alzheimer’s disease (27, 28).Very recently, NECAB1/2 were shown to have complementary expression patterns in mouse hippocampus at the mRNA and protein levels, whereas NECAB3 is broadly distributed in the hippocampus (29). NECAB1-expressing cells were seen throughout the cell-sparse layers of Ammon’s horn and the hilus of the dentate gyrus. In contrast, NECAB2 is enriched in pyramidal cells of the CA2 region. A minority of NECAB1⁺ neurons were GABAergic yet did not coexpress PV, CB, or CR (29).Here, we investigated the expression of NECAB1/2 in mouse DRGs and spinal cord using quantitative PCR (qPCR), immunohistochemistry (also combined with CLARITY) (30), and Western blotting. We compared the distribution of NECABs with that of the four CaBPs restricted to neurons, PV, CB, CR, or Scgn. NECAB⁺ neurons in the spinal dorsal horn were phenotyped using transgenic mice harboring genetic markers for excitatory [vesicular glutamate transporter 2 (VGLUT2)] (31) or inhibitory [glutamate decarboxylase 67 (GAD67)] (32) cell identities. Finally, the effect of peripheral nerve injury was analyzed.     

Placement of pacemaker leads via the extrathoracic subclavian vein guided by fluoroscopy and venography in the oblique projection

Blind needle puncture of the subclavian vein, which is the standard method used for insertion of pancemaker electrodes, causes an increased risk of lead fractures due to entrapment of the lead by the costoclavicular ligament and/or subclavius muscle. The extrathoracic lead insertion technique was developed to prevent such lead fractures. The present study was performed to evaluate the usefulness of extrathoracic subclavian vein puncture under the guidance of both fluoroscopy and venography in the oblique beam projection. Pacemaker leads were implanted in ten patients under the guidance of both fluoroscopy and venography in the ipsilateral anterior oblique projection. The angle of projection was set as large as possible between 35° and 45°. The needle was held parallel to the X-ray angle of incidence and inserted toward the first rib, then withdrawn until the tip entered the subclavian vein. This modified method of pacemaker implantation was successful in all patients, with no complications during the follow-up period ranging from 4 to 19 months. It also prevented pneumothorax and lead entrapment in soft tissue associated with the clavicle that might be caused by the conventional technique.     

电针对糖尿病胃轻瘫大鼠胃电活动与延髓神经元和星状胶质细胞可塑性的影响

[目的]观察电针对糖尿病胃轻瘫（DGP）大鼠胃电活动与延髓迷走孤束复合体（VSC）内神经元和星状胶质细胞可塑性变化的影响。[方法]实验大鼠分为空白对照（空白）组、DGP模型（模型）组、模型加电针足三里穴（足三里）组和模型加电针三阴交穴（三阴交）组。模型制备采用腹腔注射5％四氧嘧啶和熟地灌胃诱导的方法。实验3周后记录大鼠的胃电活动,取延髓进行抗Fos蛋白和抗胶质原纤维酸性蛋白（GFAP）的免疫组化染色。[结果]与空白组比较,模型组的胃电平均频率和振幅明显降低。而足三里组和三阴交组的平均频率和振幅较模型组明显升高（P〈O．01,〈0．05）;以足三里组升高更明显,与三阴交组比较差异有统计学意义（P〈0．05）。除空白组外,Fos阳性神经元、GFAP阳性星状胶质细胞集中表达于VSC。以模型组的Fos和GFAP表达最高,而足三里组与三阴交组的表达较模型组明显减少,且它们之间差异有统计学意义（P〈0．01）。[结论]针刺可改善糖尿病胃运动功能障碍,延髓VSC内免疫阳性神经元和胶质细胞可能参与了此调节作用。     

芍药苷通过抑制氧化应激及凋亡保护高糖诱导大鼠皮质神经元损伤

目的探讨芍药苷对高糖诱导大鼠皮质神经元氧化应激及凋亡的保护作用。方法从新生SD乳鼠大脑皮质分离和培养神经元,4 d后分为对照组、高糖组(50 mmol/L高糖)、低、中和高浓度组(2.5、5.0、10.0μmol/L芍药苷预处理再加50 mmol/L高糖)。采用细胞增殖活力检测试剂观察细胞活力,用吸光度(A)值表示;检测超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性;通过2′,7′-二氢二氯荧光素二乙酸酯荧光探针检测活性氧水平;Hoechst 33342染色检测细胞凋亡;免疫荧光染色测定细胞线粒体膜电位(MMP);Western blot测定Bcl-2、Bax、裂解的半胱氨酸天冬氨酸蛋白酶3(cleaved-Caspase3)蛋白表达。结果与对照组比较,高糖组A值明显降低(0.452±0.505 vs 0.985±1.010,P<0.01)。与高糖组比较,低浓度组A值无明显变化(P>0.05),中和高浓度组A值增高(0.648±0.714和0.782±0.805 vs 0.452±0.505,P<0.05,P<0.01);SOD、CAT活性增加,活性氧水平降...     

Biocompatibility and Surface Properties of TiO2 Thin Films Deposited by DC Magnetron Sputtering

We present the study of the biocompatibility and surface properties of titanium dioxide (TiO₂) thin films deposited by direct current magnetron sputtering. These films are deposited on a quartz substrate at room temperature and annealed with different temperatures (100, 300, 500, 800 and 1100 °C). The biocompatibility of the TiO₂ thin films is analyzed using primary cultures of dorsal root ganglion (DRG) of Wistar rats, whose neurons are incubated on the TiO₂ thin films and on a control substrate during 18 to 24 h. These neurons are activated by electrical stimuli and its ionic currents and action potential activity recorded. Through X-ray diffraction (XRD), the surface of TiO₂ thin films showed a good quality, homogeneity and roughness. The XRD results showed the anatase to rutile phase transition in TiO₂ thin films at temperatures between 500 and 1100 °C. This phase had a grain size from 15 to 38 nm, which allowed a suitable structural and crystal phase stability of the TiO₂ thin films for low and high temperature. The biocompatibility experiments of these films indicated that they were appropriated for culture of living neurons which displayed normal electrical behavior.     

Pain attacks in non-complicated and complicated gallstone disease have a characteristic pattern and are accompanied by dyspepsia in most patients: The results of a prospective study

Objective. The cardinal indication for surgical treatment of gallstones is pain attacks. However, following cholecystectomy, 20% of patients remain symptomatic. It is unclear to what extent post-cholecystectomy symptoms can be ascribed to persistence of preoperative symptoms or to new pathology. The pain and digestive pattern in gallstone patients has not been defined in a recent setting with ultrasonography as the diagnostic method. The aim of this study was to characterize a pain pattern that is typical for gallstone disease and to describe the extent of associated dyspepsia. Material and methods. A total of 220 patients with symptomatic gallstone disease including complicated disease (acute cholecystitis and common bile duct stones) were interviewed using detailed questionnaires to disclose pain patterns and symptoms of indigestion. Results. All patients had pain in the right upper quadrant (RUQ) including the upper midline epigastrium. The pain was localized to the right subcostal area in 20% and to the upper epigastrium in 14%, and in the rest (66%) it was more evenly distributed. An area of maximal pain could be defined in 90%. Maximal pain was located under the costal arch in 51% of patients and in the epigastrium in 41%, but in 3% behind the sternum and in 5% in the back. The pain was referred to the back in 63% of the patients. The mean visual analogue scale (VAS) score was very high: 90 mm on a 0–100 scale. A pattern of incipient or low-grade warning pain with a subsequent relatively steady state until subsiding in the same fashion was present in 90% of the patients. An urge to walk around was experienced by 71%. Pain attacks usually occurred in the late evening or at night (77%), with 85% of the attacks lasting for more than one hour and almost never less than half an hour. Sixty-six percent of the patients were intolerant to at least one kind of food, but only 48% to fatty foods. Symptoms of functional indigestion (gastroesophageal reflux, dyspepsia or irritable bowel symptoms) were seen in the vast majority in association with attacks. Conclusions. Gallstone-associated pain follows a certain pattern in the majority of patients. The pain is located in a defined area with a point of maximum intensity, is usually referred, and occurs mainly at night with duration of more than one hour. The majority of patients experience functional indigestion, mainly of the reflux type or dyspepsia.     

Verification of the membrane hypothesis of aging on the identified giant neurons of the snail Lymnaea stagnalis L. (Gastropoda, Pulmonata) by a combined application of intracellular electrophysiology and X-ray microanalysis

The validity of the membrane hypothesis of aging (Zs.-Nagy, 1978) was tested on identified giant neurons of the snail Lymnaea stagnalis L. by using a combination of intracellular microelectrophysiology and X-ray microanalysis of the intracellular water and electrolyte concentrations on the very same cells. The snails were taken from an inbred stock and divided into young, adult and old age groups (3, 12 and 24 mth, respectively). The giant neuron called LPa-2 from the left parietal ganglion was selected for the studies. The resting potential of the cell membrane was recorded by means of intracellular microelectrode technique. The very same cells were then explored by freeze fracture and analyzed by an energy dispersive bulk specimen method of X-ray microanalysis. The resting membrane potential displayed an age-dependent hyperpolarization, the intracellular water content decreased considerably and the intracellular potassium concentration increased almost 90% by old age. The relative passive permeability ratio for potassium (PK) and chloride (PCl) was calculated from the measured data by means of the Goldman-Hodgkin-Katz equation. Such calculations revealed that PK decreases nearly 50% with age causing the increase of the intracellular potassium content, and this is accompanied also by a significant decrease of the PCl. The results support the validity of the membrane hypothesis of aging and are in agreement with the general knowledge regarding the electrophysiological behaviour of the giant neurons of Gastropode snails.