Predictor role of heart rate variability in subarachnoid hemorrhage: A systematic review.

Background- Subarachnoid hemorrhage (SAH) is one of the most devastating diseases with a high rate of morbidity and mortality. The heart rate variability (HRV) is a non-invasive method of monitoring various components of the autonomic nervous system activity that can be utilized to delineate autonomic dysfunctions associated with various physiological and pathological conditions. The reliability of HRV as a predictor of clinical outcome in aneurysmal subarachnoid hemorrhage (aSAH) is not yet well investigated in literature. Methods- A systematic review and in depth analysis of 10 articles on early HRV changes in SAH patients was performed. Results- This systematic review demonstrates a correlation between early changes in HRV indices (time and frequency domain) and the development of neuro-cardiogenic complications and poor neurologic outcome in patients with SAH. Conclusions- A correlation between absolute values or changes of the LF/HF ratio and neurologic and cardiovascular complications was found in multiple studies. Because of significant limitations of included studies, a large prospective study with proper handling of confounders is needed to generate high-quality recommendations regarding HRV as a predictor of post SAH complications and poor neurologic outcome.

Risk Factors and Characteristics of Intraoperative Seizures During Awake Craniotomy: A Retrospective Cohort Study of 562 Consecutive Patients With a Space-occupying Brain Lesion.

INTRODUCTION: Intraoperative seizures (IOSs) during awake craniotomy (AC) are associated with significant morbidity. The reported incidence of IOS is between 3% and 30%. The aim of this study was to identify risk factors for IOS during AC for elective resection or biopsy of a space-occupying brain lesion. METHODS: In this retrospective study, we reviewed the records of all awake craniotomies performed by a single neurosurgeon at a single university hospital between July 2006 and December 2018. IOS was defined as a clinically apparent seizure that occurred in the operating room and was documented in the medical records. Explanatory variables were chosen based on previously published literature on risk factors for IOS. RESULTS: Five hundred and sixty-two patients had a total of 607 AC procedures during the study period; 581 cases with complete anesthesia records were included in analysis. Twenty-nine (5.0%) IOS events were reported during 29 (5%) awake craniotomies. Most seizures (27/29; 93%) were focal in nature and did not limit planned intraoperative stimulation mapping. Variables associated with IOS at a univariate P -value <0.1 (frontal location of tumor, preoperative radiotherapy, preoperative use of antiepileptic drugs, intraoperative use of dexmedetomidine, and intraoperative stimulation mapping) were included in a multivariable logistic regression. Frontal location of tumor (adjusted odds ratio: 5.68, 95% confidence interval: 2.11-15.30) and intraoperative dexmedetomidine use (adjusted odds ratio: 2.724, 95% confidence interval: 1.24-6.00) were independently associated with IOS in the multivariable analysis. CONCLUSIONS: This study identified a low incidence (5%) of IOS during AC. The association between dexmedetomidine and IOS should be further studied in randomized trials as this is a modifiable risk factor.

Benzodiazepine Sedation and Postoperative Neurological Deficits after Awake Craniotomy for Brain Tumor - An Exploratory Retrospective Cohort Study.

An awake craniotomy is a common neurosurgical procedure for excising brain tumor(s) located near or in eloquent areas. The use of benzodiazepine (BZD) for sedation in some patients with neuropathological conditions (e.g., stroke, brain tumors) has been previously linked with re-appearance of neurological deficits including limb incoordination, ataxia, and motor weakness, resulting in complications for the patient along with procedural challenges. Whether or not these findings can be extrapolated to patients undergoing brain tumor resection is largely unknown. The current work primarily sought to compare neurological outcome(s) in the immediate postoperative period between BZD-free and BZD-based sedation techniques in patients undergoing awake craniotomy. Using a database composed of awake craniotomies conducted within a single center and by a single surgeon, patients were retrospectively classified based on midazolam administration into BZD-free sedation (n=125) and BZD-based sedation (n=416) groups. Patients from each group were matched based on age, sex, tumor location, tumor grade, preoperative neurological deficits, non-operative BZD use, and Karnofsky Performance Scale scores, resulting in 108 patients within each group. Postoperative neurological deficits were recorded. Logistic regression analyses were conducted comparing postoperative neurological deficits between the matched groups. Postoperative neurological deficits were more prevalent within the BZD-based sedation group compared to the BZD-free sedation group (adjusted odds ratio (aOR)=1.903, 95% CI=1.018-3.560, p=0.044). In addition, subgroup analysis of the matched cohort showed a relationship between preoperative neurological symptoms and postoperative neurological deficits in the BZD-based sedation group (aOR=3.756, 95% CI=1.390-10.147, p=0.009). Our findings support the notion that the increased incidence of postoperative neurological deficits with BZD sedation may in part be related to the unmasking of preoperative neurological deficits. Further studies are required to confirm this phenomenon.

Preclinical assessment of onabotulinumtoxinA for the treatment of mild traumatic brain injury-related acute and persistent post-traumatic headache.

OBJECTIVE: Investigation of onabotulinumtoxinA in a murine model of acute and persistent post-traumatic headache. METHODS: Mild traumatic brain injury was induced with a weight drop method. Periorbital and hindpaw cutaneous allodynia were measured for 14 days. Mice were then exposed to bright light stress and allodynia was reassessed. OnabotulinumtoxinA (0.5 U) was injected subcutaneously over the cranial sutures at different post-injury time points. RESULTS: After milt traumatic brain injury, mice exhibited periorbital and hindpaw allodynia that lasted for approximately 14 days. Allodynia could be reinstated on days 14-67 by exposure to stress only in previously injured mice. OnabotulinumtoxinA administration at 2 h after mild traumatic brain injury fully blocked both transient acute and stress-induced allodynia up to day 67. When administered 72 h post-mild traumatic brain injury, onabotulinumtoxinA reversed acute allodynia, but only partially prevented stress-induced allodynia. OnabotulinumtoxinA administration at day 12, when initial allodynia was largely resolved, produced incomplete and transient prevention of stress-induced allodynia. The degree of acute allodynia correlated positively with subsequent stress-induced allodynia. CONCLUSION: Mild traumatic brain injury induced transient headache-like pain followed by long lasting sensitization and persistent vulnerability to a normally innocuous stress stimulus, respectively modeling acute and persistent post-traumatic headache.. Administration of onabotulinumtoxinA following the resolution of acute post-traumatic headache diminished persistent post-traumatic headache but the effects were transient, suggesting that underlying persistent mild traumatic brain injury-induced maladaptations were not reversed. In contrast, early onabotulinumtoxinA administration fully blocked both acute post-traumatic headache as well as the transition to persistent post-traumatic headache suggesting prevention of neural adaptations that promote vulnerability to headache-like pain. Additionally, the degree of acute post-traumatic headache was predictive of risk of persistent post-traumatic headache.

Technical Considerations in Awake Craniotomy with Cortical and Subcortical Motor Mapping in Preadolescents: Pushing the Envelope.

INTRODUCTION: Unlike adult gliomas, the utility of combined application of awake anesthesia and intraoperative neurophysiological monitoring (IONM) for maximal safe resection in eloquent region gliomas (ERG) has not been established for pediatric population while it remains unexplored in preadolescents (below 11 years old). CASE PRESENTATION: We report 2 cases of awake craniotomy with IONM in an 8 and 9 year old for safe maximal resection of ERG. In both the cases, repeated preoperative visits of the operating room was performed to familiarize and educate the children about intraoperative communication, comfortable positioning, and neurological assessment. Under conscious sedation protocol, cortical and subcortical mapping, and electrocorticography, gross total resection was achieved. In both the cases, there were no postoperative neurodeficits or perioperative complications. CONCLUSION: Our 2 cases illustrate the first instance of successful use of awake IONM for maximal safe resection of ERG in preadolescent age-group. We believe, with proper preoperative planning and careful titration of anesthetics, it is safe and feasible. The blanket notion that preadolescent age-group should be excluded from awake mapping needs to be challenged, rather curated on a case basis.

PU-91 drug rescues human age-related macular degeneration RPE cells; implications for AMD therapeutics.

Since mitochondrial dysfunction is implicated in the pathogenesis of AMD, this study is based on the premise that repurposing of mitochondria-stabilizing FDA-approved drugs such as PU-91, might rescue AMD RPE cells from AMD mitochondria-induced damage. The PU-91 drug upregulates PGC-1α which is a critical regulator of mitochondrial biogenesis. Herein, we tested the therapeutic potential of PU-91 drug and examined the additive effects of treatment with PU-91 and esterase inhibitors i.e., EI-12 and EI-78, using the in vitro transmitochondrial AMD cell model. This model was created by fusing platelets obtained from AMD patients with Rho0 i.e., mitochondria-deficient, ARPE-19 cell lines. The resulting AMD RPE cell lines have identical nuclei but differ in their mitochondrial DNA content, which is derived from individual AMD patients. Briefly, we report significant improvement in cell survival, mitochondrial health, and antioxidant potential in PU-91-treated AMD RPE cells compared to their untreated counterparts. In conclusion, this study identifies PU 91 as a therapeutic candidate drug for AMD and repurposing of PU-91 will be a smoother transition from lab bench to clinic since the pharmacological profiles of PU-91 have been examined already.

Awake microvascular decompression with fat-teflon sandwich technique: Clinical implications of a novel approach for cranial nerve neuralgias.

Re-appearance of trigeminal neuralgia (TN) pain following microvascular decompression (MVD) is a challenging issue. A selective ablation with MVD provides the best response in such recurrences. The absence of intra-operative indicator for immediate correction of sub-optimal decompression is the primary factor for failure. We analysed the effectiveness and safety of awake MVD in minimizing failure, by tailoring the procedure according to intra-operative response with re-exploration or additional procedure like internal neurolysis in the same setting, especially in patients without vascular compression and those unfit for General Anesthesia (GA). The prospective study from June 2016 to June 2017 includes one glossopharyngeal neuralgia (GPN) and 6 trigeminal neuralgia (TN). Five cases responded with immediate complete pain relief but in 2 cases, incomplete pain relief resulted in alteration of intraoperative decision. In one case, a partial pain relief, mandated an additional internal neurolysis in the same setting, resulting in complete pain relief while in the other, re-exploration revealed a hidden venous conflict, not identified on MRI following which an additional IN was performed. All cases were followed up with BNI PIS for a minimum of one year without recurrence. Awake MVD is safe and reliable intraoperative neurophysiological prognostic marker of immediate pain relief and provides a window for an immediate correction of sub-optimal decompression with Internal Neurolysis when needed, in the same setting, especially in neuroimaging negative and elderly cases unfit for GA. It has the potential to reduce the rate of re-intervention and increase the overall effectiveness of MVD by specifically ameliorating the pain burden and quality of life.

Chronic nicotine improves cognitive and social impairment in mice overexpressing wild type α-synuclein.

In addition to dopaminergic and motor deficits, patients with Parkinson's disease (PD) suffer from non-motor symptoms, including early cognitive and social impairment, that do not respond well to dopaminergic therapy. Cholinergic deficits may contribute to these problems, but cholinesterase inhibitors have limited efficacy. Mice over-expressing α-synuclein, a protein critically associated with PD, show deficits in cognitive and social interaction tests, as well as a decrease in cortical acetylcholine. We have evaluated the effects of chronic administration of nicotine in mice over-expressing wild type human α-synuclein under the Thy1-promoter (Thy1-aSyn mice). Nicotine was administered subcutaneously by osmotic minipump for 6 months from 2 to 8 months of age at 0.4 mg/kg/h and 2.0 mg/kg/h. The higher dose was toxic in the Thy1-aSyn mice, but the low dose was well tolerated and both doses ameliorated cognitive impairment in Y-maze performance after 5 months of treatment. In a separate cohort of Thy1-aSyn mice, nicotine was administered at the lower dose for one month beginning at 5 months of age. This treatment partially eliminated the cognitive deficit in novel object recognition and social impairment. In contrast, chronic nicotine did not improve motor deficits after 2, 4 or 6 months of treatment, nor modified α-synuclein aggregation, tyrosine hydroxylase immunostaining, synaptic and dendritic markers, or microglial activation in Thy1-aSyn mice. These results suggest that cognitive and social impairment in synucleinopathies like PD may result from deficits in cholinergic neurotransmission and may benefit from chronic administration of nicotinic agonists.

A Molecular Tweezer Ameliorates Motor Deficits in Mice Overexpressing α-Synuclein.

Aberrant accumulation and self-assembly of α-synuclein are tightly linked to several neurodegenerative diseases called synucleinopathies, including idiopathic Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Deposition of fibrillar α-synuclein as insoluble inclusions in affected brain cells is a pathological hallmark of synucleinopathies. However, water-soluble α-synuclein oligomers may be the actual culprits causing neuronal dysfunction and degeneration in synucleinopathies. Accordingly, therapeutic approaches targeting the toxic α-synuclein assemblies are attractive for these incurable disorders. The "molecular tweezer" CLR01 selectively remodels abnormal protein self-assembly through reversible binding to Lys residues. Here, we treated young male mice overexpressing human wild-type α-synuclein under control of the Thy-1 promoter (Thy1-aSyn mice) with CLR01 and examined motor behavior and α-synuclein in the brain. Intracerebroventricular administration of CLR01 for 28 days to the mice improved motor dysfunction in the challenging beam test and caused a significant decrease of buffer-soluble α-synuclein in the striatum. Proteinase-K-resistant, insoluble α-synuclein deposits remained unchanged in the substantia nigra, whereas levels of diffuse cytoplasmic α-synuclein in dopaminergic neurons increased in mice receiving CLR01 compared with vehicle. More moderate improvement of motor deficits was also achieved by subcutaneous administration of CLR01, in 2/5 trials of the challenging beam test and in the pole test, which requires balance and coordination. The data support further development of molecular tweezers as therapeutic agents for synucleinopathies.

Targeting Microglial Activation States as a Therapeutic Avenue in Parkinson's Disease.

Parkinson's disease (PD) is a chronic and progressive disorder characterized neuropathologically by loss of dopamine neurons in the substantia nigra, intracellular proteinaceous inclusions, reduction of dopaminergic terminals in the striatum, and increased neuroinflammatory cells. The consequent reduction of dopamine in the basal ganglia results in the classical parkinsonian motor phenotype. A growing body of evidence suggest that neuroinflammation mediated by microglia, the resident macrophage-like immune cells in the brain, play a contributory role in PD pathogenesis. Microglia participate in both physiological and pathological conditions. In the former, microglia restore the integrity of the central nervous system and, in the latter, they promote disease progression. Microglia acquire different activation states to modulate these cellular functions. Upon activation to the M1 phenotype, microglia elaborate pro-inflammatory cytokines and neurotoxic molecules promoting inflammation and cytotoxic responses. In contrast, when adopting the M2 phenotype microglia secrete anti-inflammatory gene products and trophic factors that promote repair, regeneration, and restore homeostasis. Relatively little is known about the different microglial activation states in PD and a better understanding is essential for developing putative neuroprotective agents. Targeting microglial activation states by suppressing their deleterious pro-inflammatory neurotoxicity and/or simultaneously enhancing their beneficial anti-inflammatory protective functions appear as a valid therapeutic approach for PD treatment. In this review, we summarize microglial functions and, their dual neurotoxic and neuroprotective role in PD. We also review molecules that modulate microglial activation states as a therapeutic option for PD treatment.

Endovascular pulmonary artery inflatable balloon-induced hypotension: A novel technique for clipping giant intracranial aneurysms.

Management of giant intracranial aneurysms presents unique challenges to the neurosurgical team. Various techniques such as adenosine-induced hypotension, rapid ventricular pacing, and inducing deep hypothermia are described in the literature to effect circulatory arrest for the successful obliteration of giant aneurysms. We describe a novel technique of induced hypotension for clipping a giant aneurysm by using an inflatable balloon across the main pulmonary artery with a successful outcome. This technique has not been described earlier in the literature.

Umbelliferone and esculetin protect against N-nitrosodiethylamine-induced hepatotoxicity in rats.

N-nitrosodiethylamine (NDEA), a nitrosamine compound, is known to cause liver damage through the generation of reactive oxygen species (ROS), resulting in oxidative damage to macromolecules such as DNA, and the consequent development of cancer. The present study examines the protective effects of two antioxidant coumarin compounds umbelliferone (Umb) and esculetin (Esc) against NDEA-induced hepatotoxicity when administered in the diet to male Wistar rats. The results show that treatment with Umb (0.5% w/w) and Esc (0.5% w/w) in the diet for 7 days significantly attenuates NDEA-induced liver damage, lowering serum alanine transaminase (ALT) levels, decreasing hepatic lipid peroxidation, and restoring total glutathione levels. To investigate the mechanism for the observed protective effect, the levels of the key protective enzymes NAD(P)H: quinone oxidoreductase 1 (NQO1), heme oxygenase (HO1), and glutathione S-transferase Pi (GSTP1) were measured by Western blotting following Umb and Esc administration. The results showed that Umb and Esc administration significantly increased the expression of NQO1 by 3.6- and 2.7-fold, HO1 by 2.7- and 3.2-fold, and GSTP1 by 2.8- and 3.2-fold, respectively. In conclusion, Umb and Esc are capable of protecting liver from NDEA-induced hepatotoxicity, and this is associated with the induction of protective enzymes.

Region specific mitochondrial impairment in mice with widespread overexpression of alpha-synuclein.

Parkinson's disease (PD) is characterized by the progressive degeneration of nigrostriatal dopaminergic neurons leading to motor deficits. The mechanisms underlying the preferential vulnerability of nigrostriatal dopaminergic neurons in PD remain poorly understood. Recent evidence supports a role for mitochondrial dysfunction and increased oxidative stress in PD pathogenesis. Genetic and pathological studies also point to alpha-synuclein as a critical factor in both familial and sporadic forms of the disease; alpha-synuclein pathology affects mitochondrial function but is widespread in PD brain, raising the question of its role in the greater vulnerability of nigrostriatal neurons in PD. We have examined mitochondrial function and oxidative damage in mice overexpressing human wild type alpha-synuclein broadly throughout the nervous system under the Thy1 promoter (Thy1-aSyn mice) between 4 and 8months of age. Similar levels of alpha-synuclein accumulation in mitochondria were detected in the ventral midbrain, striatum and cortex of Thy1-aSyn mice. However, analysis of mitochondrial respiration using Seahorse XF analyzer showed defects in mitochondrial respiratory complexes I, II, IV and V specifically in the midbrain, and IV and V in the striatum, of Thy1-aSyn mice compared to wild type littermates; mitochondrial complex I activity assay by ELISA confirmed a 40% inhibition specifically in the ventral midbrain. Mitochondrial dysfunction can contribute to oxidative stress and we observed a 40% increase in 4-hydroxynenal and 2-fold increase in malondialdehyde levels, indicative of a high level of lipid peroxidation, specifically in the ventral midbrain of Thy1-aSyn mice. The levels of peroxiredoxin 2, a neuronal antioxidant enzyme that is involved in removal of H2O2 and other toxic peroxides were decreased in the midbrain whereas its oxidized form increased 4-fold, suggesting that antioxidant defenses were compromised in this region. In contrast, peroxiredoxin 2 increased in the striatum and cortex, which may contribute to their protection in the presence of high levels of alpha-synuclein. Thus, in mice over-expressing alpha-synuclein, mitochondrial dysfunction occurred preferentially in nigrostriatal dopaminergic neurons many months before striatal dopamine loss occurs at 14months of age. This may contribute to a higher level of oxidative stress that overwhelms antioxidant defense in these neurons, leading to their increased vulnerability in PD.

Mitochondrial dysfunction and oxidative stress in Parkinson's disease.

Parkinson's disease (PD) is a movement disorder that is characterized by the progressive degeneration of dopaminergic neurons in substantia nigra pars compacta resulting in dopamine deficiency in the striatum. Although majority of the PD cases are sporadic several genetic mutations have also been linked to the disease thus providing new opportunities to study the pathology of the illness. Studies in humans and various animal models of PD reveal that mitochondrial dysfunction might be a defect that occurs early in PD pathogenesis and appears to be a widespread feature in both sporadic and monogenic forms of PD. The general mitochondrial abnormalities linked with the disease include mitochondrial electron transport chain impairment, alterations in mitochondrial morphology and dynamics, mitochondrial DNA mutations and anomaly in calcium homeostasis. Mitochondria are vital organelles with multiple functions and their dysfunction can lead to a decline in energy production, generation of reactive oxygen species and induction of stress-induced apoptosis. In this review, we give an outline of mitochondrial functions that are affected in the pathogenesis of sporadic and familial PD, and hence provide insights that might be valuable for focused future research to exploit possible mitochondrial targets for neuroprotective interventions in PD.

Neuroprotective effects of umbelliferone and esculetin in a mouse model of Parkinson's disease.

The production of reactive oxygen species and mitochondrial dysfunction in the brain are both associated with the progression of several neurodegenerative diseases, including Parkinson's disease. These characteristics are also observed when rodents are exposed to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a compound that causes nigrostriatal dopaminergic neurotoxicity and that has been used previously for assessing the effectiveness of neuroprotective agents. In this study, the neuroprotective effects of two coumarins, umbelliferone and esculetin, against MPTP-induced neurotoxicity were examined in C57BL/6J mice. The results show that dietary administration of umbelliferone and esculetin significantly attenuated MPTP-induced neurotoxicity in the substantia nigra pars compacta but not striatum, as measured by tyrosine hydroxylase staining. Both coumarins also prevented an MPTP-induced increase in nitrosative stress as measured by 3-nitrotyrosine immunoreactivity and also maintained glutathione levels in MPTP-exposed mice as well as in cell lines exposed to the metabolite 1-methyl-4-phenylpyridinium. Umbelliferone and esculetin also prevented MPTP-dependent caspase 3 activation, an indicator of apoptosis, but did not inhibit monoamine oxidase activity. This is the first time that the neuroprotective capabilities of these coumarins have been demonstrated, and the results indicate that umbelliferone and esculetin can protect against MPTP-induced neurotoxicity in the mouse. These compounds can cross the blood-brain barrier, so their effectiveness indicates that they have the potential to protect in neurodegenerative disease such as Parkinson's disease.

A progressive mouse model of Parkinson's disease: the Thy1-aSyn ("Line 61") mice.

Identification of mutations that cause rare familial forms of Parkinson's disease (PD) and subsequent studies of genetic risk factors for sporadic PD have led to an improved understanding of the pathological mechanisms that may cause nonfamilial PD. In particular, genetic and pathological studies strongly suggest that alpha-synuclein, albeit very rarely mutated in PD patients, plays a critical role in the vast majority of individuals with the sporadic form of the disease. We have extensively characterized a mouse model over-expressing full-length, human, wild-type alpha-synuclein under the Thy-1 promoter. We have also shown that this model reproduces many features of sporadic PD, including progressive changes in dopamine release and striatal content, alpha-synuclein pathology, deficits in motor and nonmotor functions that are affected in pre-manifest and manifest phases of PD, inflammation, and biochemical and molecular changes similar to those observed in PD. Preclinical studies have already demonstrated improvement with promising new drugs in this model, which provides an opportunity to test novel neuroprotective strategies during different phases of the disorder using endpoint measures with high power to detect drug effects.

Esculetin-induced protection of human hepatoma HepG2 cells against hydrogen peroxide is associated with the Nrf2-dependent induction of the NAD(P)H: Quinone oxidoreductase 1 gene.

Esculetin (6,7-dihydroxy coumarin), is a potent antioxidant that is present in several plant species. The aim of this study was to investigate the mechanism of protection of esculetin in human hepatoma HepG2 cells against reactive oxygen species (ROS) induced by hydrogen peroxide. Cell viability, cell integrity, intracellular glutathione levels, generation of reactive oxygen species and expression of antioxidant enzymes were used as markers to measure cellular oxidative stress and response to ROS. The protective effect of esculetin was compared to a well-characterized chemoprotective compound quercetin. Pre-treatment of HepG2 cells with sub-lethal (10-25 µM) esculetin for 8h prevented cell death and maintained cell integrity following exposure to 0.9 mM hydrogen peroxide. An increase in the generation of ROS following hydrogen peroxide treatment was significantly attenuated by 8h pre-treatment with esculetin. In addition, esculetin ameliorated the decrease in intracellular glutathione caused by hydrogen peroxide exposure. Moreover, treatment with 25 µM esculetin for 8h increased the expression of NAD(P)H: quinone oxidoreductase (NQO1) at both protein and mRNA levels significantly, by 12-fold and 15-fold, respectively. Esculetin treatment also increased nuclear accumulation of Nrf2 by 8-fold indicating that increased NQO1 expression is Nrf2-mediated. These results indicate that esculetin protects human hepatoma HepG2 cells from hydrogen peroxide induced oxidative injury and that this protection is provided through the induction of protective enzymes as part of an adaptive response mediated by Nrf2 nuclear accumulation.