The repeated cross fostering protocol as a mouse model of panic disorder: suggestions for new treatments from behavioral and molecular characterization

Panic disorder (PD) is a common psychiatric illness with highly stereotyped symptoms including a sense of shortness of breath or feelings of suffocation. PD is characterized by spontaneous and recurrent panic attacks (PAs) that consist of incapacitating periods of acute-onset respiratory, cardiovascular, gastrointestinal, autonomic and cognitive symptoms. According to the DSM-5, recurrent panic attacks in PD are categorized as being either spontaneous (unexpected) or cued (expected). Accumulating evidence suggests that spontaneous PAs may be provoked by interoceptive sensory triggers caused by fluctuations in the internal homeostatic milieu. An important internal homeostatic trigger for the genesis of PAs, supported by an emerging body of work, is acid–base imbalance and associated pH chemosensory mechanisms. Largely founded on panic provocation studies with agents promoting homeostatic pH imbalance (e.g. sodium lactate or CO2) and related to the false suffocation alarm theory the role of acid–base and chemosensory systems in panic provides strong scientific insights on the genesis of PAs. Heightened sensitivity to carbon dioxide (CO2) is an established biological correlate of PD. Indeed inhaled CO2 triggers PAs in most individuals with PD but only a minority of unaffected controls. More recently the acid sensing ion channel-1 (ASIC1) has been proposed as a candidate gene responsible for these phenomena. Indeed this channel is expressed in central nervous system and in particular in amygdala, brainstem, structures involved in chemosensory detection and breathing. According to twin studies, shared genetic determinants appear to be the major underlying cause of the developmental of adult PD and of altered sensitivity to CO2. Moreover, in addition to genetic determinants, environmental risk factors affect the liability to these traits, and several life events that influence the susceptibility to PD also predict heightened CO2 reactivity. There is evidence that genetic and environmental determinants interact to influence human responses to CO2 suggesting also epigenetic mechanisms to underlie the development of PD. Valuable animal models for PD are lacking due to the difficulty to measure “panic” in animals. Indeed models of PD used in pre-clinical research measure the defensive behaviors showed by the animals in response to a real aversive stimulus and not spontaneously and/or in the absence of real dangerous situation, as in PD. Not being able to interview the animal, about its symptoms, such as fear of dying or going crazy as in human PD patients, CO2 hypersensitivity, observed in patients with panic and their unaffected relatives, represents a valid endophenotype to model this disorder in animals. PD is a chronic disorder with variable course and treatments available until now are not specific and are often only modestly efficacious. Typical pharmacologic treatments are antidepressants (SSRI) or anxiolytics (benzodiazepines). An alternative strategy is psychotherapy (cognitive behavioral therapy) and often patients are treated with a combination of psycho- and pharmaco-therapies. In some cases therapies (e.g., benzodiazepines) may be associated with treatment-specific side effects or risks such as sedation or the risk of dependence or tolerance. For these reasons is important to find a therapy specific for PD, possibly with the fewest side effects. The aims of my PhD Thesis were: 1) to validate the RCF protocol in mice as a useful manipulation procedure affecting individual emotionality, different from the classical maternal separation (Handling), usually applied in rodents, to evaluate the effects of an early adverse environment. I evaluated the short and long-term effects of these early manipulations on several behavioral, molecular and physiological parameters (mother-pups interaction; stress response; emotionality; CO2 panic-related response; gluco- and mineral-corticoid receptors mRNA expression; etc.) 2) to analyze possible molecular mechanisms underlying the panic-related CO2 hypersensitivity showed by RCF animals and evaluate different pharmacological treatments (chloridiazepoxide, chlorogenic acid and amiloride) able to recover the normal respiratory response to hypercapnia, on the basis of molecular suggestions 3) to verify the cognitive capability of RCF animals trough learning tests (such as active avoidance test and novel recognition test) and investigate the capability of exposure to 6% CO2 on animals’ behavioral conditioning, in RCF and Control subjects. Indeed, humans with PD show behavioral conditioning to panic attacks and develop PA also in absence of unconditioned stimulus. 4) to investigate whether the CO2 hypersensitivity showed by RCF animals was a transgenerational transmissible trait. First of all, results reported in this study suggest that the behavioral and physiological phenotypes observed during development and adulthood depend on characteristics and timings of early adversities capable of activate different biological processes. Reasonably, the response of the animal to the early manipulations is different and aimed at maximizing individual fitness: the early environment could exert its programming role during this developmental plastic period, through specific epigenetic modifications. Short, even if repeated, separations from the mother (Handling protocol) induce habituation to a relatively low stressing environment, enhancing the capability of the subject to face new stressful situations. By contrast, the disruption of the infant attachment bond (RCF protocol) is associated to a modification in the respiratory response to high CO2 in breathing air, an endophenotype these animals share with PD patients. The disruption of infant-mother bond in RCF animals suggested by the enhanced separation anxiety at 8 days age supports the relation between SAD and PD already suggested in literature. In addition the CO2 reactivity showed by these animals represents a useful tool to study PD in pre-clinical research. Molecular alterations found in RCF animals (experiment 2a) supported the involvement of acid-base balance dysregulation in development of CO2 hypersensitivity. Indeed RCF animals showed a higher expression in ASIC1 gene that codifies for acid sensing ion channels. These channels are sensitive to lower levels of pH being able to detect changes in CO2 concentration in the body and adjust the respiratory function to receive enough O2 not to compromise biological processes. Molecular investigations in addition revealed alterations in GABAergic transmission in RCF animals supporting the idea of an involvement of this neurotransmitter in the development of PD. RCF animals showed an increased expression of Dbi which is an inhibitor of GABAergic transmission. These molecular findings have provided indications suggesting that a possible rescue treatment for PD patients should consist in reducing CO2 hypersensitivity. Lowering of this increased respiratory response to modest increase in CO2 could reduce the negative feeling associated to condition, reducing the conditioning potentiality that favor the development of panic disorder, after repeated panic attacks. The use of benzodiazepine such as chlordiazepoxide was able to restore the normal respiratory response to CO2 as well, giving pharmacological validation to RCF model. However, benzodiazepines have several contraindications, especially for chronic treatments and their sedative effect should also be taken into consideration. Even if I only present few data on the effects of chlorogenic acid and amiloride on RCF animals, I think these results are very interesting and need further and deeper evaluation. Both these compounds interacted with the pH sensitive channels (asics) and their administration was able to restore the respiratory response observed in control animals. It is well known, that panic attacks are able to condition behaviors of PD patients. They indeed tend to avoid situations and places similar to those where a panic attack previously occurred. Similarly RCF animals showed, in experiment 3, behavioral conditioning to the situation previously paired with CO2 (tone exposure). It should be now explored whether RCF animals generalize the conditioned fear, suggesting how an initial panic attack can evolve into panic disorder in humans. Finally RCF model demonstrated a transgenerational transmission of the respiratory endophenotype (experiment 4) supporting the hypothesis of gene-enviroment interplay role to predisposition to panic disorder (Spatola et al., 2011). The epigenetic mechanisms responsible for this trans-generational transmission are under investigation as well as possible strategies to prevent this phenomenon. In conclusion, the Repeated Cross-Fostering protocol seems a valid mouse model of Panic Disorder in humans: RCF mice show typical features of this disorder such as separation anxiety during childhood, CO2 hypersensitivity and CO2 conditioned and avoidance behaviors. Acid sensing ion channels are interesting molecular markers which can be used as new targets for pharmacological treatments and can help to explain hyper-responsiveness to CO2 in PD patients as well

Early handling and repeated cross-fostering have opposite effect on mouse emotionality

Early life events have a crucial role in programming the individual phenotype and exposure to traumatic experiences during infancy can increase later risk for a variety of neuropsychiatric conditions, including mood and anxiety disorders. Animal models of postnatal stress have been developed in rodents to explore molecular mechanisms responsible for the observed short and long lasting neurobiological effects of such manipulations. The main aim of this study was to compare the behavioral and hormonal phenotype of young and adult animals exposed to different postnatal treatments. Outbred mice were exposed to (i) the classical Handling protocol (H: 15 min-day of separation from the mother from day 1 to 14 of life) or to (ii) a Repeated Cross-Fostering protocol (RCF: adoption of litters from day 1 to 4 of life by different dams). Handled mice received more maternal care in infancy and showed the already described reduced emotionality at adulthood. Repeated cross fostered animals did not differ for maternal care received, but showed enhanced sensitivity to separation from the mother in infancy and altered respiratory response to 6% CO2 in breathing air in comparison with controls. Abnormal respiratory responses to hypercapnia are commonly found among humans with panic disorders (PD), and point to RCF-induced instability of the early environment as a valid developmental model for PD. The comparisons between short-and long-term effects of postnatal handling vs. RCF indicate that different types of early adversities are associated with different behavioral profiles, and evoke psychopathologies that can be distinguished according to the neurobiological systems disrupted by early-life manipulation

Mouse model of panic disorder: Vulnerability to early environmental instability is strain-dependent.

AbstractEarly life experiences and genetic background shape phenotypic variation. Several mouse models based on early treatments have evaluated short‐ and long‐term phenotypic alterations and explored their molecular mechanisms. The instability of maternal cues was used to model human separation anxiety in outbred mice, one of the etiopathogenetic factors that predict panic disorder (PD). Application of the repeated cross‐fostering (RCF) protocol to inbred strains (C57 and DBA) allowed us to measure differential responses to the same experimental manipulation. Ultrasounds emitted during isolation indicated that after RCF, pups from both strains lose their ability to be comforted by nest cues, but the frequency modulation of separation calls increased in RCF‐C57 and decreased in RCF‐DBA mice. No strain‐specific difference in olfactory ability explained these responses in RCF‐exposed mice. Rather, disruption of the infant‐mother bond may differentially affect separation calls in the two strains. Moreover, the RCF‐associated increased respiratory response to hypercapnia–an endophenotype of human PD documented among mice outbred strains–was replicated in the C57 strain only. We suggest that RCF‐induced instability of the early environment affects emotionality and respiratory physiology differentially, depending on pups' genetic background. These strain‐specific responses provide a lead to understand differential vulnerability to emotional disorders

CD34 selected cells for the treatment of poor graft function after allogeneic stem cell transplantation.

Abstract Poor graft function (PGF) is characterized by pancytopenia and a hypoplastic marrow, with complete donor chimerism, usually without severe graft-versus-host disease (GVHD). We report 41 patients with PGF, treated with granulocyte colony-stimulating factor–mobilized CD34 selected cells, at a median interval from transplant of 140 days, without conditioning and without GVHD prophylaxis. Donors were HLA matched siblings (n = 12), unrelated donors (n = 18), or mismatched family members (n = 11). The median number of infused CD34 + cells was 3.4 × 10 6 /kg. The rate of trilineage recovery was 75%: 83% for HLA matched siblings and 72% for unrelated and mismatched family members ( P = .3). The cumulative incidence of acute grade II GVHD was 15%, and no patient developed de novo chronic GVHD. The actuarial 3-year survival is 63%: 76% and 25% for patients with or without trilineage recovery. These data confirm the role of CD34 + selected cells from the same donor in the treatment of PGF and warrant the request for a second donation also when the donor is unrelated

Histone Modifications in a Mouse Model of Early Adversities and Panic Disorder: Role for Asic1 and Neurodevelopmental Genes

Hyperventilation following transient, CO2-induced acidosis is ubiquitous in mammals and heritable. In humans, respiratory and emotional hypersensitivity to CO2 marks separation anxiety and panic disorders, and is enhanced by early-life adversities. Mice exposed to the repeated cross-fostering paradigm (RCF) of interference with maternal environment show heightened separation anxiety and hyperventilation to 6% CO2-enriched air. Gene-environment interactions affect CO2 hypersensitivity in both humans and mice. We therefore hypothesised that epigenetic modifications and increased expression of genes involved in pH-detection could explain these relationships. Medullae oblongata of RCF- and normally-reared female outbred mice were assessed by ChIP-seq for H3Ac, H3K4me3, H3K27me3 histone modifications, and by SAGE for differential gene expression. Integration of multiple experiments by network analysis revealed an active component of 148 genes pointing to the mTOR signalling pathway and nociception. Among these genes, Asic1 showed heightened mRNA expression, coherent with RCF-mice’s respiratory hypersensitivity to CO2 and altered nociception. Functional enrichment and mRNA transcript analyses yielded a consistent picture of enhancement for several genes affecting chemoception, neurodevelopment, and emotionality. Particularly, results with Asic1 support recent human findings with panic and CO2 responses, and provide new perspectives on how early adversities and genes interplay to affect key components of panic and related disorders

Post-Transplant Nivolumab Plus Unselected Autologous Lymphocytes in Refractory Hodgkin Lymphoma: A Feasible and Promising Salvage Therapy Associated With Expansion and Maturation of NK Cells

Immune checkpoint inhibitors (CI) have demonstrated clinical activity in Hodgkin Lymphoma (HL) patients relapsing after autologous stem cell transplantation (ASCT), although only 20% complete response (CR) rate was observed. The efficacy of CI is strictly related to the host immune competence, which is impaired in heavily pre-treated HL patients. Here, we aimed to enhance the activity of early post-ASCT CI (nivolumab) administration with the infusion of autologous lymphocytes (ALI). Twelve patients with relapse/refractory (R/R) HL (median age 28.5 years; range 18-65), underwent lymphocyte apheresis after first line chemotherapy and then proceeded to salvage therapy. Subsequently, 9 patients with progressive disease at ASCT received early post-transplant CI supported with four ALI, whereas 3 responding patients received ALI alone, as a control cohort. No severe adverse events were recorded. HL-treated patients achieved negative PET scan CR and 8 are alive and disease-free after a median follow-up of 28 months. Four patients underwent subsequent allogeneic SCT. Phenotypic analysis of circulating cells showed a faster expansion of highly differentiated NK cells in ALI plus nivolumab-treated patients as compared to control patients. Our data show anti-tumor activity with good tolerability of ALI + CI for R/R HL and suggest that this setting may accelerate NK cell development/maturation and favor the expansion of the "adaptive" NK cell compartment in patients with HCMV seropositivity, in the absence of HCMV reactivation

Phenotype and genotype of 87 patients with Mowat-Wilson syndrome and recommendations for care

Mowat-Wilson syndrome (MWS) is a rare intellectual disability/multiple congenital anomalies syndrome caused by heterozygous mutation of the ZEB2 gene. It is generally underestimated because its rarity and phenotypic variability sometimes make it difficult to recognize. Here, we aimed to better delineate the phenotype, natural history, and genotype-phenotype correlations of MWS.MethodsIn a collaborative study, we analyzed clinical data for 87 patients with molecularly confirmed diagnosis. We described the prevalence of all clinical aspects, including attainment of neurodevelopmental milestones, and compared the data with the various types of underlying ZEB2 pathogenic variations.ResultsAll anthropometric, somatic, and behavioral features reported here outline a variable but highly consistent phenotype. By presenting the most comprehensive evaluation of MWS to date, we define its clinical evolution occurring with age and derive suggestions for patient management. Furthermore, we observe that its severity correlates with the kind of ZEB2 variation involved, ranging from ZEB2 locus deletions, associated with severe phenotypes, to rare nonmissense intragenic mutations predicted to preserve some ZEB2 protein functionality, accompanying milder clinical presentations.ConclusionKnowledge of the phenotypic spectrum of MWS and its correlation with the genotype will improve its detection rate and the prediction of its features, thus improving patient care.GENETICS in MEDICINE advance online publication, 4 January 2018; doi:10.1038/gim.2017.221