MORPHOMETRIC STUDY OF ISOLATED DOGIEL TYPE II CELLS OF THE INTERMUSCULAR PLEXUS

Aim of the study was to calculate the morphometric parameters of isolated Dogiel type II cells from the intermuscular plexus of the small intestine. Materials and methods: 3D models of the oval-shaped Dogiel type II cells from the intermuscular plexus of a rat (n = 1) were constructed and studied. Neurocytes were identified by the impregnation method on the frontal and horizontal sections of the wall of the small intestine. Results: The results showed that the total number of the nodes in the virtual model was 36534, and the mesh elements — 156595. The resulting 3D model of the cell and nucleus was reduced 900 times to obtain a threedimensional cell and nucleus with absolute dimensions, with a ratio of 1:1 to their true size. The volume of Dogiel type II cell was 2785.11 μm³, the volume of the nucleus was 647.7 μm³ and the volume of its perikaryon was 2785.11 μm³. Conclusion: Dogiel type II cells from the intermuscular plexus of the rat small intestine has an ovoid shaped threedimensional structure. These cells are flattened in transverse direction and elongated in longitudinal direction.

Developmental Changes in Peripherin-eGFP Expression in Spiral Ganglion Neurons

The two types of spiral ganglion neurons (SGNs), types I and II, innervate inner hair cells and outer hair cells, respectively, within the mammalian cochlea and send another process back to cochlear nuclei in the hindbrain. Studying these two neuronal types has been made easier with the identification of unique molecular markers. One of these markers, peripherin, was shown using antibodies to be present in all SGNs initially but becomes specific to type II SGNs during maturation. We used mice with fluorescently labeled peripherin (Prph-eGFP) to examine peripherin expression in SGNs during development and in aged mice. Using these mice, we confirm the initial expression of Prph-eGFP in both types I and II neurons and eventual restriction to only type II perikarya shortly after birth. However, while Prph-eGFP is uniquely expressed within type II cell bodies by P8, both types I and II peripheral and central processes continue to express Prph-eGFP for some time before becoming downregulated. Only at P30 was there selective type II Prph-eGFP expression in central but not peripheral processes. By 9 months, only the type II cell bodies and more distal central processes retain Prph-eGFP expression. Our results show that Prph-eGFP is a reliable marker for type II SGN cell bodies beyond P8; however, it is not generally a suitable marker for type II processes, except for central processes beyond P30. How the changes in Prph-eGFP expression relate to subsequent protein expression remains to be explored.

MicroRNA 29 Family Serves a Key Role in Lung Development and the Prevention of Lung Pathology

Even with remarkable advances in the care of preterm infants, chronic lung disease in the form of bronchopulmonary dysplasia (BPD) continues to be a significant pathologic consequence of prematurity. BPD is caused by the interruption of physiologic lung development and exposure of the immature newborn lung to high O2 tensions. BPD is characterized by a simplified alveolar structure, arrested lung growth, impaired vascular development and lung fibrosis (1). To identify effective treatment strategies for BPD, it is important to understand the molecular mechanisms underlying this disorder. MicroRNAs (miRNA, miR) are known to regulate growth, development and repair of the developing lung; whereas, dysregulation of miRNA expression has been associated with pulmonary disease. Specifically, members of the miR-29 family have been linked to pulmonary cancers, fibrosis, and BPD. Previous studies from our laboratory indicate that developmental induction of miR-29 expression in the fetal lung near term serves a key role in promoting surfactant-producing type II cell differentiation and function through repression of TGF-β2 signaling (2). To understand the role of miR-29 in protection against BPD, in the present study, we created mice in which the entire miR-29 family (miR-29a/b1 and miR-29b2/c) (miR-29 dKO) was disrupted. Upon exposure of miR-29 dKO and WT neonatal mice to hyperoxia (95% O2) for 5 days immediately after birth, expression levels of the proinflammatory cytokines and chemokines, IL-1 β, TGF-β1, CXCL2 and IL-6, were significantly increased in the lungs of miR-29 dKO mice, compared to WT. Furthermore, lungs of miR-29 dKO adult mice manifested increased expression of their direct targets, TGF-β2 and TGF-β3. This was associated with increased collagen deposition, as evidenced by enhanced trichrome staining, suggesting the development of lung fibrosis. HDAC4 (a direct target of miR-29) and proinflammatory TNF-α, which have been implicated in pulmonary fibrosis, also were upregulated in the lungs of miR-29 dKO mice. Overall, our studies suggest a key role of the miR-29 family and its targets in prevention of inflammatory and profibrotic signaling in the neonatal lung leading to lung pathology. Supported by: NIH R01-HL050022 (C.R.M.) References: (1)Michael, Zoe, et al. “Bronchopulmonary dysplasia: an update of current pharmacologic therapies and new approaches.” Clinical Medicine Insights: Pediatrics 12 (2018): 1179556518817322.(2)Guo, Wei, Houda Benlhabib, and Carole R. Mendelson. “The microRNA 29 family promotes type II cell differentiation in developing lung.” Molecular and cellular biology 36.16 (2016): 2141-2141.

Covid-19 Interstitial Pneumonia: Histological and Immunohistochemical Features on Cryobiopsies

Background: The pathogenetic steps leading to Covid-19 interstitial pneumonia remain to be clarified. Most postmortem studies to date reveal diffuse alveolar damage as the most relevant histologic pattern. Antemortem lung biopsy may however provide more precise data regarding the earlier stages of the disease, providing a basis for novel treatment approaches. Objectives: To ascertain the morphological and immunohistochemical features of lung samples obtained in patients with moderate Covid-19 pneumonia. Methods: Transbronchial lung cryobiopsy was carried out in 12 Covid-19 patients within 20 days of symptom onset. Results: Histopathologic changes included spots of patchy acute lung injury with alveolar type II cell hyperplasia, with no evidence of hyaline membranes. Strong nuclear expression of phosphorylated STAT3 was observed in >50% of AECII. Interalveolar capillaries showed enlarged lumen and were in part arranged in superposed rows. Pulmonary venules were characterized by luminal enlargement, thickened walls, and perivascular CD4+ T-cell infiltration. A strong nuclear expression of phosphorylated STAT3, associated with PD-L1 and IDO expression, was observed in endothelial cells of venules and interstitial capillaries. Alveolar spaces macrophages exhibited a peculiar phenotype (CD68, CD11c, CD14, CD205, CD206, CD123/IL3AR, and PD-L1). Conclusions: Morphologically distinct features were identified in early stages of Covid-19 pneumonia, with epithelial and endothelial cell abnormalities different from either classical interstitial lung diseases or diffuse alveolar damage. Alveolar type II cell hyperplasia was a prominent event in the majority of cases. Inflammatory cells expressed peculiar phenotypes. No evidence of hyaline membranes and endothelial changes characterized by IDO expression might in part explain the compliance and the characteristic pulmonary vasoplegia observed in less-advanced Covid-19 pneumonia.

Expanding Role of Dopaminergic Inhibition in Hypercapnic Responses of Cultured Rat Carotid Body Cells: Involvement of Type II Glial Cells

Dopamine (DA) is a well-studied neurochemical in the mammalian carotid body (CB), a chemosensory organ involved in O2 and CO2/H+ homeostasis. DA released from receptor (type I) cells during chemostimulation is predominantly inhibitory, acting via pre- and post-synaptic dopamine D2 receptors (D2R) on type I cells and afferent (petrosal) terminals respectively. By contrast, co-released ATP is excitatory at postsynaptic P2X2/3R, though paracrine P2Y2R activation of neighboring glial-like type II cells may boost further ATP release. Here, we tested the hypothesis that DA may also inhibit type II cell function. When applied alone, DA (10 μM) had negligible effects on basal [Ca2+]i in isolated rat type II cells. However, DA strongly inhibited [Ca2+]i elevations (Δ[Ca2+]i) evoked by the P2Y2R agonist UTP (100 μM), an effect opposed by the D2/3R antagonist, sulpiride (1–10 μM). As expected, acute hypercapnia (10% CO2; pH 7.4), or high K+ (30 mM) caused Δ[Ca2+]i in type I cells. However, these stimuli sometimes triggered a secondary, delayed Δ[Ca2+]i in nearby type II cells, attributable to crosstalk involving ATP-P2Y2R interactions. Interestingly sulpiride, or DA store-depletion using reserpine, potentiated both the frequency and magnitude of the secondary Δ[Ca2+]i in type II cells. In functional CB-petrosal neuron cocultures, sulpiride potentiated hypercapnia-induced Δ[Ca2+]i in type I cells, type II cells, and petrosal neurons. Moreover, stimulation of type II cells with UTP could directly evoke Δ[Ca2+]i in nearby petrosal neurons. Thus, dopaminergic inhibition of purinergic signalling in type II cells may help control the integrated sensory output of the CB during hypercapnia.