In vivo identification of angle dysgenesis and its relation to genetic markers associated with glaucoma using artificial intelligence.

PURPOSE: To predict the presence of angle dysgenesis on anterior-segment optical coherence tomography (ADoA) by using deep learning (DL) and to correlate ADoA with mutations in known glaucoma genes. PARTICIPANTS: In total, 800 high-definition anterior-segment optical coherence tomography (AS-OCT) images were included, of which 340 images were used to build the machine learning (ML) model. Images used to build the ML model included 170 scans of primary congenital glaucoma (16 patients), juvenile-onset open-angle glaucoma (62 patients), and adult-onset primary open-angle glaucoma eyes (37 patients); the rest were controls (n = 85). The genetic validation dataset consisted of another 393 images of patients with known mutations that were compared with 320 images of healthy controls. METHODS: ADoA was defined as the absence of Schlemm's canal, the presence of hyperreflectivity over the region of the trabecular meshwork, or a hyperreflective membrane. DL was used to classify a given AS-OCT image as either having angle dysgenesis or not. ADoA was then specifically looked for on AS-OCT images of patients with mutations in the known genes for glaucoma. RESULTS: The final prediction, which was a consensus-based outcome from the three optimized DL models, had an accuracy of >95%, a specificity of >97%, and a sensitivity of >96% in detecting ADoA in the internal test dataset. Among the patients with known gene mutations, ( MYOC, CYP1B1, FOXC1, and LTBP2 ) ADoA was observed among all the patients in the majority of the images, compared to only 5% of the healthy controls. CONCLUSION: ADoA can be objectively identified using models built with DL.

Digenic Inheritance in Juvenile Open-Angle Glaucoma.

Juvenile open-angle glaucoma (JOAG) is an uncommon subset of primary glaucoma with an onset before the age of 40 years. In this case report, we describe the cosegregation of MYOC , p.Pro370Leu and LTBP2 , p.Pro432Leu mutations in a family with JOAG. The family with autosomal dominant JOAG belonged to Northern India. The samples of proband and her parents were evaluated by whole exome sequencing. Sanger sequencing was conducted in all the study participants to check the mutations identified. Both MYOC and LTBP2 mutations were found to cosegregate in affected individuals leading to a severe JOAG phenotype, thereby suggesting a digenic inheritance of MYOC with LTBP2 in this family.

A new association of PAX6 variation with Juvenile onset open angle glaucoma.

Mutations in the PAX6 gene are generally associated with aniridia. We describe a family with Juvenile onset open angle glaucoma (JOAG), where one of the two children had JOAG and the other Juvenile ocular hypertension. Whole exome sequencing was performed for the parents and their two affected children where the proband and her sibling were detected to have a de novo PAX6 gene variant in the absence of aniridia. All previously described gene mutations for glaucoma were looked for in the family. The potential pathogenicity of the identified variants was assessed by determining their frequency in large public exome databases; as well as using the current ACMG guidelines. The same heterozygous variant at NM_000280.6:c.1124 C > A; p. Pro375Gln in the PAX6 gene was detected in the proband and her affected brother. The variant has been described in aniridia patients before and has been shown to cause a weaker DNA binding using functional studies. This report expands the phenotypic spectrum of the PAX6 gene to include Juvenile onset open angle glaucoma.

Association of EFEMP1 with juvenile-onset open angle glaucoma in a patient with concomitant COL11A1-related Stickler syndrome.

BACKGROUND: Juvenile onset open-angle glaucoma is described as a primary open-angle glaucoma, with an age of onset before 40 years. These patients have a higher prevalence of myopia. PURPOSE: We describe the phenotype of juvenile onset open-angle glaucoma in a patient with a rare variant in EFEMP1 gene, who was also detected to have Stickler syndrome(STL). METHODS: Whole exome sequencing (WES) was undertaken in 40 unrelated families where the proband had juvenile onset open-angle glaucoma (JOAG). RESULTS: Out of these, eight were autosomal dominant, while the rest did not have any other affected first-degree relative. Out of the 8 autosomal dominant JOAG families, MYOC mutations were detected in 3(37.5%) and LTBP2 in 1(12.5%). One family (12.5%) had a rare EFEMP1 sequence variant in both affected father and daughter. The daughter also had high myopia and a pathogenic COL11A1 sequence variant that led to a coincidental diagnosis of STL in her. CONCLUSIONS: This is a rare association of EFEMP1 and COL11A1 sequence variants in a JOAG patient with STL. The study also reiterates the association of JOAG with EFEMP1, which should be looked for, especially in families with autosomal dominant JOAG.

Distribution of TGFBI variants in patients with early onset glaucoma.

Purpose: To describe a novel association of TGFBI variants with congenital glaucoma in a family with GAPO (growth retardation, alopecia, pseudoanodontia, and progressive optic atrophy) syndrome, as well as among other unrelated cases of juvenile onset open-angle glaucoma (JOAG) and primary congenital glaucoma (PCG). Methods: This study of one family of GAPO with congenital glaucoma and three unrelated patients with JOAG analyzed a common link to glaucoma pathogenesis. Three girls with GAPO syndrome born to consanguineous parents in a multi-generation consanguineous family were identified. Two of the girls had congenital glaucoma in both eyes, while the elder sibling (a 10-year-old female) had features of GAPO syndrome without glaucoma. Results: A genetic evaluation using whole exome sequencing revealed a novel homozygous ANTXR1 mutation in all three affected siblings with GAPO. No other mutations were detected in the genes associated with glaucoma. A rare missense variant in the TGFBI gene was shared in the two siblings with congenital glaucoma and GAPO syndrome. We found three other unrelated patients with JOAG and one patient with primary congenital glaucoma with no known glaucoma causing gene mutations, but having four different missense variants in the TGFBI gene. One of these patients with JOAG had familial granular corneal dystrophy. Molecular dynamic simulations of TGFBI and 3-D structural models of three of its variants showed significant alterations that could influence TGFBI protein function. Conclusions: The possibility that variations in the TGFBI gene could have a possible role in the pathogenesis of congenital and juvenile onset open-angle glaucomas needs further evaluation.

Posterior corneal morphological changes in primary congenital glaucoma.

Purpose: To compare posterior corneal morphology between older treated and younger untreated children with primary congenital glaucoma (PCG) using anterior segment optical coherence tomography (ASOCT) and intraoperative OCT (iOCT), respectively. Methods: In this comparative study, ASOCT of older PCG children were compared with iOCT of younger untreated PCG patients. Differences between the two groups with respect to posterior corneal morphology were studied. Results: Observed morphological patterns within posterior cornea in older treated (age: 72-300 months) children (87 eyes) included Descemet's membrane (DM) excrescences (70%), thickened DM (35%), intracameral twin protuberances (92%), and DM detachment (26%). Changes within pre-Descemet's layer (PDL) (28%) included thickening, breaks, and detachments. Extent of Haab's striae was associated with thickness of DM/PDL complex (P = 0.008) when analyzed in the treated group. In contrast, in the untreated group (n = 53 eyes, age 1-63 months), posterior corneal changes were limited to diffuse hyper-reflectivity of the DM/PDL complex, with absence of DM tears. Conclusion: Posterior cornea thickens and Haab's striae become more circumscribed in eyes of older treated children compared to untreated PCG eyes, probably reflecting a healing response of posterior cornea over time.

Exome sequencing identifies procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 mutations in primary congenital and juvenile glaucoma.

PURPOSE: To report the association of procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) mutations with bilateral primary congenital glaucoma (PCG) in monozygotic twins and with nondominant juvenile-onset primary open-angle glaucoma (JOAG). METHODS: We utilized family-based whole-exome sequencing to detect disease-causing mutations in a pair of monozygotic twins with de-novo PCG and compared its existence in 50 nonfamilial cases of JOAG and 30 healthy controls. To validate the identified mutations, direct Sanger sequencing was performed. For further evaluation of gene expression in the ocular tissues, we performed whole-mount in situ hybridization in zebrafish embryos. RESULTS: We identified a novel missense mutation (c.1925A>G, p.Tyr642Cys) in the PLOD2 gene in the monozygotic twin pair with PCG and another missense mutation (c.1880G>A, p.Arg627Gln) in one JOAG patient. Both mutations identified were heterozygous. Neither the parents of the twins nor the parents of the JOAG patient harbored the mutation and it was probably a de-novo change. The zebrafish in situ hybridization revealed expression of the PLOD2 gene during embryogenesis of the eye. CONCLUSION: We observed an association of PLOD2 mutations with PCG and with nonfamilial JOAG. This new gene needs to be further investigated for its role in pathways associated with glaucoma pathogenesis.

Myopia and glaucoma progression among patients with juvenile onset open angle glaucoma: A retrospective follow up study.

OBJECTIVE: To identify the risk factors for glaucoma progression, especially the association with myopia, among treated juvenile open angle glaucoma (JOAG) patients. METHODS: Glaucomatous progression was analysed in the eyes of JOAG patients with at least 5-years of follow up in this retrospective study. Baseline variables such as age, inheritance pattern, baseline intraocular pressure (IOP), baseline central corneal thickness, visual acuity, baseline refractive error, spherical equivalent (SE) and duration of follow-up were noted. Stereoparametric global trend analysis and Moorfields Regression Analysis on confocal scanning laser ophthalmoscopy were used to detect progression. Variables associated with glaucoma progression, with respect to progressors (PG) and non-progressors (NPG) were analysed. Since both eyes of a patient were taken for analysis, a generalised estimating equation method was used to correct the bias. RESULTS: Among 74 eyes (37 subjects), glaucoma progression was noted in 11 eyes (14.9%) of 8 patients, with a median time to progression of 7.4 years (range 5-15.5 years). For myopes (SE ≤ -1.00 D), glaucoma progression was 18 times more likely than mild and no myopes (>-1DS) (p = 0.03, 95% CI: 1.14, 217.44). The prevalence of myopia in the JOAG, PG and NPG cohorts was 70.3%, 87.5%, and 65.5%, respectively. Myopia progression was noted at follow up in 70% patients. One-unit increase in baseline vertical cup disc ratio, 1 mmHg increase in IOP fluctuations and 1 dB year-1 depression of visual field were associated with 0.44, 0.06 and 0.07 D year-1 increases in the rate of myopia progression, respectively. CONCLUSIONS: JOAG progressors had a greater baseline myopic refraction and a faster myopia development over time. The development of myopia in JOAG eyes could be an indicator of glaucoma progression, and hence progressing myopic (≤-1 D) JOAG patients should be followed up more rigorously.

The mutational spectrum of Myocilin gene among familial versus sporadic cases of Juvenile onset open angle glaucoma.

PURPOSE: Juvenile onset primary open angle glaucoma (JOAG) is a rare disorder associated with high IOP and progressive optic neuropathy in patients diagnosed before the age of 40 years. While in some populations it has primarily an autosomal dominant pattern of inheritance, in others it occurs in a primarily sporadic form. The main aim of the study was to assess the relative prevalence of Myocilin (MYOC) mutations in familial versus sporadic cases of JOAG. METHODS: We screened 92 unrelated (sporadic) JOAG patients, and 22 affected families (70 affected members and 36 unaffected) for variations in the MYOC gene. We also analyzed the clinical features associated with these variations. RESULTS: Three coding sequence variants were identified as mutations causing JOAG. Four families segregated distinct mutations at Gly367Arg, and two families at Gln337Arg, while only two sporadic JOAG cases harbored MYOC mutations (Gly367Arg and Gln48His). The frequency of MYOC mutations in familial cases (27%) was significantly higher than in sporadic JOAG cases (2%); p = 0.001. A 90% penetrance for the Gly367Arg variant was seen by the age of 40 years in our patients. Characteristic allele signatures, indicative of specific founder effects, were not observed for the Gly367Arg mutation that was looked for in 12 patients among 2 geographically close families, which harbored this mutation. CONCLUSION: Our data demonstrated that genetic screening for MYOC mutations should be focused toward cases with familial rather than sporadically occurring JOAG.

Differences in outflow channels between two eyes of unilateral primary congenital glaucoma.

PURPOSE: Primary congenital glaucoma (PCG) occurs in only one eye in some patients. We aimed to characterize anatomical features of the angle and Schlemm's canal (SC) in vivo among fellow eyes of patients with unilateral primary congenital glaucoma. METHODS: Both eyes of 33 children with unilateral PCG and 30 healthy, age-matched children, old enough to co-operate were analysed using high-resolution anterior segment spectral domain (SD) OCT. Subgroup analysis was done for the presence/absence of angle dysgenesis as defined by the presence of abnormal tissue/hyper-reflective membrane within angle recess and/or the absence of SC. Other anatomical landmarks differentiating the fellow eyes from eyes with glaucoma were also evaluated and compared with healthy subjects. RESULTS: The presence of abnormal tissue at the angle and/or a hyper-reflective membranous structure covering the meshwork was seen in all affected PCG eyes (100%) and in 21 (63%) unaffected fellow eyes; p = 0.001. The SC could be seen in 8 (24%) affected in comparison with 29 (88%) fellow unaffected eyes; p = 0.001. The ASOCT scans of 54 (90%) healthy eyes and 3 (9%) fellow PCG eyes revealed a direct communication of anterior portion of the SC with the anterior chamber. Among the fellow eyes, a communication of the supraciliary space with anterior chamber could be discerned in 26 eyes (79%). CONCLUSIONS: Despite angle dysgenesis, outflow channels such as the uveoscleral or a direct communication of SC with the anterior chamber play a role in preventing the development of glaucoma in fellow eyes of unilateral PCG.

Classifying juvenile onset primary open angle glaucoma using cluster analysis.

AIM: To classify unrelated patients with juvenile onset primary open angle glaucoma (JOAG) into clinically useful phenotypes using cluster analysis. METHODS: Out of the 527 unrelated patients with JOAG, the study included 414 patients who had all the phenotypic characteristics required for the study. A cluster analysis was performed to classify the patients based on their iris and angle morphology, age of onset, highest untreated intraocular pressure (IOP), worst mean deviation and greatest vertical cup disc ratio of the worst eye. The iris features were broadly classified into three groups: those with normal iris crypts (NIC), those with prominent iris crypts (PIC) and those with absence of iris crypts. The gonio photographs were graded as normal appearing angle or those with angle dysgenesis in the form of a featureless angle, one with a high iris insertion and an angle with prominent iris processes. Using a hierarchical clustering model and a two-way cluster analysis, the distribution of clusters of JOAG was analysed to obtain a classification of JOAG subtypes. RESULTS: The four major clusters identified were: Cluster 1 with NIC and normal angles had the lowest untreated IOP and higher age of onset among all clusters. Cluster 2 with NIC and featureless angle was found to be associated with earliest age of onset. Cluster 3 had NIC and either a high iris insertion or prominent iris processes. Cluster 4 was a heterogeneous cluster with maximum number of patients in a group comprising of those with PIC and high iris insertion. CONCLUSIONS: Cluster analysis extracted four subgroups of the JOAG phenotype that have clinical and prognostic significance and can potentially be helpful while evaluating these patients in the clinics.

Long-term functional outcomes of different subtypes of primary congenital glaucoma.

AIM: To analyse long-term visual outcomes across different subtypes of primary congenital glaucoma (PCG). METHODS: Patients with PCG with a minimum of 5-year follow-up post surgery were included in the study. Snellen visual acuity recordings taken at their last follow-up were analysed. We evaluated the results using Kaplan-Meier curves to predict the probability of maintaining good vision (as defined by a visual acuity of 6/18 or better) in our patients after 30-year follow-up. The results were also analysed to determine whether there were any differences in the long-term visual acuities with time between the neonatal and infantile PCG. We also analysed the reasons for poor visual outcomes. RESULTS: We assessed a cohort of 140 patients with PCG (235 eyes) with an average follow-up of 127±62.8 months (range 60-400 months). Overall, the proportion of eyes with good visual acuity was 89 (37.9%), those with fair visual acuity between 6/60 and 6/18 was 41 (17.4%), and those with poor visual acuity (≤6/60) was 105 (44.7%). We found a significant difference (p=0.047) between neonatal and infantile patients with PCG whereby the neonatal cohort fared worse off in terms of visual morbidity. On Kaplan-Meier analysis, the cumulative probability of survival of a visual acuity of 6/18 or better was more among the infantile PCG in comparison to the neonatal PCG (p=0.039) eyes, and more among the bilateral than the unilateral affected eyes (p=0.029). Amblyopia was the most important cause for poor visual acuity as shown on a Cox proportional-hazards regression model. CONCLUSIONS: Long-term visual outcomes of infantile are better than neonatal PCG. Eyes with unilateral have worse visual outcomes compared with those with bilateral PCG because of the development of dense amblyopia.

Molecular characterization of a rare phenotype of X-linked retinoschisis with angle-closure glaucoma.

A 11-year-old boy presented with complaints of blurred vision and on evaluation was found to have X-linked retinoschisis (XLRS) with angle-closure glaucoma. Clinical and genetic evaluation of first-degree family members was done. His brother had a milder form of XLRS with shallow anterior chamber. Topical dorzolamide 2% and timolol 0.5% were used to control intraocular pressure. Genetic analysis revealed a novel three base pair deleterious mutation (c. 375_377 del AGA) in exon-5 of the RS1 gene in three members of the family.

Lasers in glaucoma.

While lasers have been used for many years for the treatment of glaucoma, proper indications and use of the procedures need to be considered before their application. This review summarizes the important laser procedures in Glaucoma.

Therapeutic potential of valproic acid in advanced glaucoma: A pilot study.

Purpose: Oral valproic acid (VPA) used as an anticonvulsant has been shown to improve contrast threshold sensitivities in patients receiving it on long-term. This study aimed to evaluate the efficacy of oral VPA in improving visual function in eyes with advanced stage glaucoma. Methods: In this prospective randomized study, 31 patients (n = 31 eyes) with advanced stage glaucoma (with an intraocular pressure <16 mmHg) in at least one eye received oral VPA 500 mg once a day for 3 months and 33 patients (n = 33 eyes) continued on glaucoma therapy. Patients were followed up at 3 and 12 months (to evaluate the legacy effect of the drug). Blood VPA concentrations were measured at 3 months. Following parameters were assessed at baseline, 3 months and 12 months: log of the minimum angle of resolution (LogMAR) visual acuity, mean deviation on visual fields, and multifocal electroretinogram (ERG). Results: Median LogMar visual acuity in the VPA group improved from 0.3 at baseline to 0.18 and 0.18 at 3 and 12 months, respectively (P < 0.01). In comparison, the median visual acuity in control group at baseline was 0.18 and showed neither worsening nor improvement over 3 and 12 months (P = 0.56). The improvement in VPA group was significant compared to the control group (P < 0.01; Wilcoxon Signed-rank test). An improvement in one line was experienced in 11 out of 31 eyes in the VPA group compared to 1 out of 33 eyes among controls (P = 0.003). No significant improvement was noted in the mean deviation, and the multifocal ERG (Latency and amplitudes) in the VPA-treated patients. The average blood VPA concentration measured at 3 months of therapy was 26 ± 8.9 µg/ml (range 8-55 µg/ml) which is much lower than that achieved during anticonvulsant therapy. None of the patients complained of any adverse effects that required stopping VPA therapy. Conclusion: A 3 months oral VPA therapy results in some improvement in visual acuity in a subgroup of eyes with advanced glaucoma and the effect was seen to persist 9 months after the drug was stopped.

Phenotypic differences between familial versus non-familial Juvenile onset open angle glaucoma patients.

AIM: To evaluate phenotypic differences among familial and non-familial JOAG patients. METHODS: First degree relatives of unrelated JOAG patients were screened for glaucoma and ocular hypertension. JOAG probands were grouped as familial or non-familial and phenotypic differences in terms of age of onset, gender, baseline untreated IOP, presence angle dysgenesis, and refractive error was compared between the two groups. RESULTS: Out of 368 unrelated JOAG patients, 134 in whom all first degree relatives had been examined were included in the study. The non-familial JOAG (n = 96) had similar age of onset as familial JOAG (n = 38); (p = 0.076) but had greater male preponderance (p = 0.046), and had the higher baseline IOP (p = 0.044) compared to familial JOAG. However, on adjustment using the Bonferroni correction, the observed differences were not found to be significant. Both groups had similar proportion of patients with angle dysgenesis (p = 0.46) and high myopia (p = 0.72). CONCLUSIONS: Non-familial JOAG were not found to be phenotypically different from the familial JOAG patients in this cohort.

Role of CYP1B1, p.E229K and p.R368H mutations among 120 families with sporadic juvenile onset open-angle glaucoma.

BACKGROUND: To determine the frequency of CYP1B1 p.E229K and p.R368H, gene mutations in a cohort of sporadic juvenile onset open-angle glaucoma (JOAG) patients and to evaluate their genotype/phenotype correlation. METHODS: Unrelated JOAG patients whose first-degree relatives had been examined and found to be unaffected were included in the study. The patients and their parents were screened for p.E229K and p.R368H mutations. The phenotypic characteristics were compared between probands carrying the mutations and those who did not carry these mutations. RESULTS: Out of 120 JOAG patients included in the study, the p.E229K mutation was seen in 9 probands (7.5%) and p.R368H in 7 (5.8%). The average age of onset of the disease (p = 0.3) and the highest untreated IOP (p = 0.4) among those carrying mutations was not significantly different from those who did not have these mutations. The proportion of probands with angle dysgenesis among those with p.E229K and p.R368H mutations was 70% (11 out of 16) in comparison to 65% (67 out of 104) of those who did not harbour these mutations (p = 0.56). Similarly, the probands with moderate to high myopia among those with p.E229K and p.R368H mutations was 20% (3 out of 16) in comparison to 18% (18 out of 104) of those who did not harbour these mutations (p = 0.59). CONCLUSION: The frequency of p.E229K and p.R368H mutations of the CYP1B1 gene is low even among sporadic JOAG patients. Moreover, there is no clinical correlation between the presence of these mutations and disease severity.

Noradrenaline from Locus Coeruleus Neurons Acts on Pedunculo-Pontine Neurons to Prevent REM Sleep and Induces Its Loss-Associated Effects in Rats.

Normally, rapid eye movement sleep (REMS) does not appear during waking or non-REMS. Isolated, independent studies showed that elevated noradrenaline (NA) levels inhibit REMS and induce REMS loss-associated cytomolecular, cytomorphological, psychosomatic changes and associated symptoms. However, the source of NA and its target in the brain for REMS regulation and function in health and diseases remained to be confirmed in vivo. Using tyrosine hydroxylase (TH)-siRNA and virus-coated TH-shRNA in normal freely moving rats, we downregulated NA synthesis in locus coeruleus (LC) REM-OFF neurons in vivo. These TH-downregulated rats showed increased REMS, which was prevented by infusing NA into the pedunculo-pontine tegmentum (PPT), the site of REM-ON neurons, normal REMS returned after recovery. Moreover, unlike normal or control-siRNA- or shRNA-injected rats, upon REMS deprivation (REMSD) TH-downregulated rat brains did not show elevated Na-K ATPase (molecular changes) expression and activity. To the best of our knowledge, these are the first in vivo findings in an animal model confirming that NA from the LC REM-OFF neurons (1) acts on the PPT REM-ON neurons to prevent appearance of REMS, and (2) are responsible for inducing REMSD-associated molecular changes and symptoms. These observations clearly show neuro-physio-chemical mechanism of why normally REMS does not appear during waking. Also, that LC neurons are the primary source of NA, which in turn causes some, if not many, REMSD-associated symptoms and behavioral changes. The findings are proof-of-principle for the first time and hold potential to be exploited for confirmation toward treating REMS disorder and amelioration of REMS loss-associated symptoms in patients.

Rapid Eye Movement Sleep Deprivation Induces Neuronal Apoptosis by Noradrenaline Acting on Alpha1 Adrenoceptor and by Triggering Mitochondrial Intrinsic Pathway.

Many neurodegenerative disorders are associated with rapid eye movement sleep (REMS) loss; however, the mechanism was unknown. As REMS loss elevates noradrenaline (NA) level in the brain as well as induces neuronal apoptosis and degeneration, in this study, we have delineated the intracellular molecular pathway involved in REMS deprivation (REMSD)-associated NA-induced neuronal apoptosis. Rats were REMS deprived for 6 days by the classical flower pot method; suitable controls were conducted and the effects on apoptosis markers evaluated. Further, the role of NA was studied by one, intraperitoneal (i.p.) injection of NA-ergic alpha1 adrenoceptor antagonist prazosin (PRZ) and two, by downregulation of NA synthesis in locus coeruleus (LC) neurons by local microinjection of tyrosine hydroxylase siRNA (TH-siRNA). Immunoblot estimates showed that the expressions of proapoptotic proteins viz. Bcl2-associated death promoter protein, apoptotic protease activating factor-1 (Apaf-1), cytochrome c, caspase9, caspase3 were elevated in the REMS-deprived rat brains, while caspase8 level remained unaffected; PRZ treatment did not allow elevation of these proapoptotic factors. Further, REMSD increased cytochrome c expression, which was prevented if the NA synthesis from the LC neurons was blocked by microinjection of TH-siRNA in vivo into the LC during REMSD in freely moving normal rats. Mitochondrial damage was re-confirmed by transmission electron microscopy, which showed distinctly swollen mitochondria with disintegrated cristae, chromosomal condensation, and clumping along the nuclear membrane, and all these changes were prevented in PRZ-treated rats. Combining findings of this study along with earlier reports, we propose that upon REMSD NA level increases in the brain as the LC, NA-ergic REM-OFF neurons do not cease firing and TH is upregulated in those neurons. This elevated NA acting on alpha1 adrenoceptors damages mitochondria causing release of cytochrome c to activate intrinsic pathway for inducing neuronal apoptosis in REMS-deprived rat brain.