Correlation Between Pathological Changes Of Olfactory Epithelium And The Occurrence And Severity Of Olfactory Disorder

The first partThe relationship between apoptosis of olfactory epithelial stem cells andinflammatory diseases leading to olfactory disordersObjectiveUse specific antibodies to label olfactory epithelial stem cells,and explore the relationship between changes in olfactory epithelial stem cells at different olfactory levels in patients with chronic rhino-sinusitis and the occurrence and development of olfactory disorders.Methods1.T&T olfactory assessment was performed on 48 patients with chronic rhinosinusitis.According to the results,the subjects were divided into five groups:normal sense of smell(3cases),mild decrease(18cases),moderate decrease(12cases).severe decline(3cases),olfactory loss group(12cases).Sign the sample written informed consent,take a piece of 0.5x0.5cm2 olfactory area and fix it with 4%paraformaldehyde.2.The normal olfactory sample was used as a control.All samples were stained with HE to evaluate the overall morphology of the olfactory epithelium(OE).The number of cells was counted by ImageJ software to calculate the cell density.Olfactory sensory neurons were labeled by OMP.Olfactory receptor neurons(ORNs),Ascl-1 labeled globose basal cells(GBCs),CK-5-labeled horizontal basal cells(HBCs)were used to observe the changes of stem cells in each group,and TUNEL was used to observe the apoptosis.ImageJ counts the number of positive cells.Results1.Olfactory normal group:(1)OE overall structure rules,surface cilia,cells in each layer are arranged neatly,basement membrane is not obvious;(2)ORNs are numerous in number,regular in shape,neatly arranged,and complete surface olfactory cilia;(3)see A small number of ORNs direct the positive signal of precursor stem cells(Ascl-1+GBCs),scattered in the lower OE;(4)HBCs are arranged neatly and the cells are in a resting state;(5)TUNEL signals are not seen in the OE layer,but scattered in the inherent layer..2.Slightly decreased olfactory group:(1)The overall structure of OE remained regular,the cells in each layer were well-arranged,the surface cilia was sparse,the basement membrane was not thickened,and the cell density was not changed significantly(2)The number of ORNs did not change significantly.However,the integrity of the surface olfactory ciliates was not as good as that of the normal group;(3)The positive signal of Ascl-1+GBCs was increased compared with the normal group;(4)The HBCs were arranged neatly and cells appeared.Activity trends;(5)The TUNEL signal appears at the OE level and is located at the top of the OE.3.Moderately decreased olfactory group:(1)The overall structure of OE remained regular,with neat rows of cells,increased damage to the surface ciliary structure,no thickening of the basement membrane,and a slight decrease in cell density;(2)Increased olfactory ciliary damage,ORNs morphological structure remains intact,but partial loss;(3)Ascl-1~+GBCs positive signal increased significantly;(4)HBCs cell activity increased significantly;(5)OE TUNEL signal increased Partially coincides with Ascl-1~+GBCs and does not coincide with HBCs.4.Severe olfactory decline group:(1)The overall structure of the OE was severely damaged,with significant medulla and cilia disappearing.The structure of the false coating was unclear,the cell loss was obvious,the basement membrane was significantly thickened,and the OE cell density decreased significantly.Intrinsic layer of blood vessels increased;(2)olfactory cilia completely disappeared,ORNs loss increased,leaving a small number of ORNs,but most of the cell structure is incomplete;(3)positive signals Ascl-1~+GBCs are almost invisible;(4)HBCs are still activated,but cell activity is not as good as before;(5)The TUNEL signal in OE increases significantly,almost distributes OE,partly overlaps with Ascl-1+GBCs,and does not coincide with HBCs.5.Olfactory loss group:(1)The basement membrane was thicker and more obvious,and the OE cell density was the lowest.Two pathological changes were observed:(1)OE structure was severely damaged,most of the OE cell structure disappeared,and 2)some respiratory epithelium appeared.(2)The olfactory cilia completely disappeared,scattered in the ORNs signal,and the cell structure was illegible;(3)There was no positive signal of Ascl-1~+GBCs;(4)The cell activity of HBCs disappeared;(5)The TUNEL signal was overlaid in the visual field.No TUNEL signal and HBCs were overlapped in the patients with respiratory epithelial metaplasia.TUNEL signals in OE severe disruptors coincided with HBCs.Conclusions1.The olfactory epithelial stem cells Ascl-1~+GBCs were compensated in the early stage of olfactory disorders,and the number of supplemental neurons was increased.At this time,the olfactory sensation did not change significantly,and the individuals were at the level of mild to moderate olfactory sensation;Existence,disease progression,apoptosis of Ascl-1~+GBCs surpasses their compensatory capacity,renders the ORNs untimely,the olfactory sensation subsides,and the loss of individual olfactory sensations to severely absent levels or even loss.2.HBCs are activated to participate in repair when OE is damaged,but when OE destruction reaches a certain level,HBCs are inhibited from activating and even induce apoptosis of HBCs.The second part Pathological changes of olfactory epithelium in OVA-induced olfactory dysfunction mouse modelObjectiveAfter the first part of the experiment,we initially concluded that the apoptosis of olfactory epithelial stem cells is closely related to the occurrence of olfactory disorders.In this section we will compare the pathological changes of the olfactory epithelium in mouse models with changes in clinical samples and analyze the feasibility of using animal models for the study of olfactory disorders.Methods1.Olfactory dysfunction was induced by ovalbumin(OVA)sensitization and intranasally induced in mice.Animal models were established: 27 SPF-grade 6-week-old male BALB/c mice were randomly selected and divided into 9 groups(n=3).Dissolve OVA in sterile normal saline to make a 100 mg/m L stock solution,then dilute the stock solution and mix thoroughly with aluminum hydroxide adjuvant in a volume ratio of 1:1 to make a suspension.Take 50 ?g of the suspension in the first place.The mice were sensitized by intraperitoneal injection on day 7,day 14,and day 14.The mice were treated with 10 ?L of the stock solution for intranasal nasal instillation on the 21 st day.The mice in the control group were given equal amounts of physiological saline(NS)to obtain the OVA instillation for 1 week(1W)./OVA)and the same time saline drip nasal control group(1W/NS);2W/OVA,2W/NS;4W/OVA,4W/NS;8W/OVA,8W/NS and untreated control mice.All mice were immersed in the omnivorous test 24 h after the last nasal instillation to assess the olfactory sensation.The olfactory information was collected and sacrificed.The skin and soft tissue of the head of the mouse were removed and fixed in 4% paraformaldehyde.2.After obtaining the mouse olfactory region specimen,perform the same experimental treatment with the first part of the clinical sample,and statistically analyze the obtained data,and compare with the results of the first part of the clinical experiment.Results1.There was no significant difference in olfactory evaluation between saline control group and control group mice;1W/OVA,2W/OVA,4W/OVA mice had negative correlation with olfactory level and exposure duration of OVA,but There was no significant decrease in the level of olfactory,and there was no significant difference in olfactory sensation between the groups.The olfactory sensation in 8W/OVA mice was significantly decreased.2.(1)The OE of 1W/OVA mice was not significantly different from the control group.The distribution of ORNs was dense,uniform,and tidy.OE showed a small number of scattered Ascl-1+GBCs signals.HBCs were arranged neatly and inactive.No significant TUNEL signal was observed(a small part of the visual field showed OE structure destruction,the number of ORNs decreased HBCs activation and TUNEL signal appeared in the OE surface)(2)2W/OVA and 1W/OVA decreased,and the structure and cell morphology were not obvious Abnormal changes,Ascl-1 + GBCs signal increase,HBCs arranged neatly,inactive,the abnormal visual field increased;(3)4W/OVA nasal tissue structure no obvious abnormal changes,Ascl-1 + GBCs signal was obvious,HBCs began Activation,abnormal visual field was significantly higher than 2W/OVA;(4)OE of 8W/OVA mice showed significant pathological changes,decreased cell density,loss of ORNs,thinning of epithelial layer,and angiogenesis in the intrinsic layer,Ascl-1+ GBCs disappeared and HBCs proliferated.Conclusions1.Mouse model of olfactory dysfunction can be established by sensitization of OVA and intranasal induction of murine nasal inflammation;2.The relationship between the pathological changes of OE and olfactory epithelial stem cells and the degree of olfactory loss in the mouse model is similar to that of the clinical samples,and can be used as a study of olfactory disorders in a certain extent.