Dr. Dan Siegel - Wheel of Awareness Meditations

Below are various versions of the Wheel of Awareness meditation Dr. Siegel has developed, from easier (beginner) to more complex and expanded (advance). He led us through this process (about 20 minutes) yesterday at the Evolution of Psychotherapy Conference - great experience, and there is clinical evidence that some of the more advanced processes are profoundly effective at improving the quality of life of the practitioner, IF the practice is done daily.

His new diagram is a quadrant model: UR = 5 Senses, which is where he begins the meditation, UL = Interoception (internal physical states) [6th sense], LL = Mental/Cognitive content, including emotions [7th sense], LR = Interconnections, connections to others, inclusive [8th sense].

The audio can be downloaded at the links below - or you can listen to them at Dr. Siegel's page (linked to below).

Wheel of Awareness

Wheel of Awareness - Introduction

October 14, 2013

In this 8 minute wheel of awareness introduction, you can hear a description of the metaphor of the wheel to illuminate the nature of consciousness and its differentiated parts including the hub of knowing, the rim of the known, and the spoke of attention.

download mp3 >> (right click on link to save file)

Wheel of Awareness - Basic

October 14, 2013
In this 25 minute wheel of awareness practice, each of the segments of the rim are explored and the practice ends with the fourth segment of our sense of connection to others.

download mp3 >> (right click on link to save file)

Wheel of Awareness - Expanded

October 14, 2013

In this 29 minute expanded wheel of awareness practice, the basic elements are included and in addition to expanded reflective elements are added: 1) Awareness of awareness with the bending of the spoke of attention back towards the hub of knowing; 2) During the fourth segment focus on our sense of connectedness, the research-proven statements of positive intentions and kindness are offered to promote self- and other-directed compassion.
 
download mp3 >> (right click on link to save file) 

Wheel of Awareness - Consolidated

October 14, 2013

This is a practice that should only be done after mastering the basic and expanded practices. This is offered by popular request for those familiar with the wheel to have a more expedited experience available for their busy lives! At least it is comprehensive and over the minimum dozen minutes some suggest is necessary for daily practice! In this 15 minute wheel of awareness practice, the breath becomes a pacer for the movement of the spoke of attention around the rim. Some people find it helpful when on the third segment of the rim to count the number of breaths by pressing on the fingers of one hand to reach five for each of the first parts of that segment, and to a count of ten for the "awareness of awareness" portion as well. When this becomes familiar to you, you can use your own timing to allow this consolidated practice without listening to an external voice.

download mp3 >> (right click on link to save file)

Daniel J. Siegel: What Is Interpersonal Neurobiology?

Since I was fortunate enough to get to see and hear Dan Siegel a couple of times this conference, I thought I would share the joy as much as possible. This podcast is the Producer's Pick this week at Sounds True - a great conversation between Tami Simon and Dr. Siegel.

Daniel J. Siegel: What Is Interpersonal Neurobiology?

By following both his scientific curiosity and his heart, Dr. Daniel Siegel began to question one of the most fundamental assumptions about human psychology and even biology: that we are an individual, separate self. Dr. Siegel discovered that our concept of “I” turns out to be more accurately a “we”—encompassing not only our own senses, brain, and awareness, but also—surprisingly enough—the world itself, including everyone around us that we interact with and merge with in every moment of our lives. In this excerpt from the audio course The Neurobiology of “We,” selected by Sounds True producer Randy Roark, Dr. Siegel explores some of the key observations that have caused us to alter the essential definition of who we are.

Download »

More from Daniel J. Siegel:

 

The Mindful Brain: The Neurobiology of Well-Being

The Neurobiology of "We" - How Relationships, the Mind, and the Brain Interact to Shape Who We Are

Practicing Mindsight

Mindfulness and the Brain: A Professional Training in the Science and Practice of Meditative Awareness (w/ Jack Kornfield)

Influence of Post-Traumatic Stress Disorder on Neuroinflammation and Cell Proliferation in a Rat Model of Traumatic Brain Injury

 

From PLoS ONE, this is an excellent overview of how post-traumatic stress disorder (PTSD) and traumatic brain injury (TBI) on neuroinflammation and neurogenesis (in a rat model). The researchers compared levels of neuroinflammation and neurogenesis between TBI alone and TBI+PTSD and found that PTSD did not exacerbate the neuropathological markers of TBI.

However,

These results indicate a progressive deterioration of the TBI brain, which, under the conditions of the present approach, was not intensified by PTSD, at least within our time window and within the examined areas of the brain. Although the PTSD manipulation employed here did not exacerbate the pathological effects of TBI, the observed long-term inflammation and suppressed cell proliferation may evolve into more severe neurodegenerative diseases and psychiatric disorders currently being recognized in traumatized TBI patients.

The long-term impact of PTSD in the brain still indicates that the inflammation and reduction of neurogenesis can cause cognitive deficits.

Full Citation: 
Acosta SA, Diamond DM, Wolfe S, Tajiri N, Shinozuka K, et al. (2013) Influence of Post-Traumatic Stress Disorder on Neuroinflammation and Cell Proliferation in a Rat Model of Traumatic Brain Injury. PLoS ONE 8(12): e81585. doi:10.1371/journal.pone.0081585

Influence of Post-Traumatic Stress Disorder on Neuroinflammation and Cell Proliferation in a Rat Model of Traumatic Brain Injury
 

Sandra A. Acosta, David M. Diamond, Steven Wolfe, Naoki Tajiri, Kazutaka Shinozuka, Hiroto Ishikawa, Diana G. Hernandez, Paul R. Sanberg, Yuji Kaneko, Cesar V. Borlongan 

Published: December 09, 2013

Abstract

Long-term consequences of traumatic brain injury (TBI) are closely associated with the development of severe psychiatric disorders, such as post-traumatic stress disorder (PTSD), yet preclinical studies on pathological changes after combined TBI with PTSD are lacking. In the present in vivo study, we assessed chronic neuroinflammation, neuronal cell loss, cell proliferation and neuronal differentiation in specific brain regions of adult Sprague-Dawley male rats following controlled cortical impact model of moderate TBI with or without exposure to PTSD. Eight weeks post-TBI, stereology-based histological analyses revealed no significant differences between sham and PTSD alone treatment across all brain regions examined, whereas significant exacerbation of OX6-positive activated microglial cells in the striatum, thalamus, and cerebral peduncle, but not cerebellum, in animals that received TBI alone and combined TBI-PTSD compared with PTSD alone and sham treatment. Additional immunohistochemical results revealed a significant loss of CA3 pyramidal neurons in the hippocampus of TBI alone and TBI-PTSD compared to PTSD alone and sham treatment. Further examination of neurogenic niches revealed a significant downregulation of Ki67-positive proliferating cells, but not DCX-positive neuronally migrating cells in the neurogenic subgranular zone and subventricular zone for both TBI alone and TBI-PTSD compared to PTSD alone and sham treatment. Comparisons of levels of neuroinflammation and neurogenesis between TBI alone and TBI+PTSD revealed that PTSD did not exacerbate the neuropathological hallmarks of TBI. These results indicate a progressive deterioration of the TBI brain, which, under the conditions of the present approach, was not intensified by PTSD, at least within our time window and within the examined areas of the brain. Although the PTSD manipulation employed here did not exacerbate the pathological effects of TBI, the observed long-term inflammation and suppressed cell proliferation may evolve into more severe neurodegenerative diseases and psychiatric disorders currently being recognized in traumatized TBI patients.

Introduction

Approximately 2 million Americans every year suffer traumatic brain injury (TBI) [1]. Due to medical advances, the mortality rate associated with TBI has declined from 24.9 per 100,000 US residents in 1979 to 17.8 per 100,000 US residents in 2007 [2], [3]. However, an estimated 90,000 survivors will experience loss of physical and cognitive functions [4]. As a consequence, there is an increase of TBI-related chronic illnesses such as memory impairments and, neuropsychological disabilities including depression, anxiety, and post-traumatic stress disorder (PTSD), which impedes quality of life and contributes to a high cost of disability annually [4], [5]. These TBI-induced neuropsychological disabilities either persist or develop late in life and may precipitate anxiety disorders and PTSD in veterans and civilians [6], [7], [8], [9]. However, there is no clear evidence on how these psychiatric morbidities interact with chronic TBI [6].

Accumulating evidence indicates TBI closely presents with neurological impairments, which progressively worsen over time, and lead to secondary injuries instigating a diffused neuroinflammatory response [1], [5], [10], [11], [12] and neurogenic alterations [13], [14], [15]. Although these early immunological and neural disturbances are becoming recognized in the laboratory, the long-term pathological consequences of TBI have remained underexplored. In particular, whether traumatic stress at the time of TBI exacerbates chronic neuroinflammation and suppressed neurogenesis is not fully understood. To this end, the present in vivo study recognized the gap in knowledge on the pathological link between TBI and PTSD, and embarked on characterizing the neuroinflammatory response, neuronal cell loss, cell proliferation and neuronal differentiation by integrating an animal model of chronic TBI with a well-established animal model of PTSD [16], [17], [18]. The emergence of PTSD as a major co-morbidity factor associated with TBI is an urgent clinical unmet need. Because TBI has become the signature wound of wars in Iraq and Afghanistan, improving the clinical outcome will most likely require treating TBI, as well as co-morbid disorders, including PTSD.

Materials and Methods

Subjects

Experimental procedures were approved by the University of South Florida Institutional Animal Care and Use Committee (IACUC). All animals were housed under ambient conditions (20°C, 50% relative humidity, and a 12-h light/dark cycle), and necessary precautions were undertaken throughout the study to minimize pain and stress associated with the experimental treatments. All studies were performed by personnel blinded to the treatment conditions.
 
TBI surgical procedures

Ten-week old Sprague–Dawley rats (n = 24) were subjected to either moderate TBI using a controlled cortical impactor (CCI) (n = 12, n = 6 TBI alone and n = 6 TBI-PTSD) or sham treatment (no TBI) (n = 6 sham surgery-no PTSD and n = 6 sham surgery-PTSD). Deep anesthesia was achieved using 1–2% isoflurane, and it was maintained using a gas mask. All animals were fixed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). After exposing the skull, the CCI rod impacted the brain at the fronto-parietal cortex (coordinates of −0.2 mm anterior and +0.2 mm lateral to the midline) with a velocity of 6.0 m/s reaching a depth of 1.0 mm below the dura and remained in the brain for 150 milliseconds. The CCI rod was angled 15° degrees vertically to maintain a perpendicular position in reference to the tangential plane of the brain curvature at the impact surface. A linear variable displacement transducer (Macrosensors, Pennsauken, NJ), which was connected to the impactor, measured the velocity and duration to verify consistency across animals. Sham control injury surgeries (i.e., uninjured animals) consisted of animals exposed to anesthesia, scalp incision, craniectomy, and suturing. An electric drill was used to performed the craniectomy of about 4 mm radius centered from bregma −0.2 anterior and +0.2 mm lateral right. A computer operated thermal blanket pad and a rectal thermometer allowed maintenance of body temperature within normal limits. All animals were closely monitored post-operatively with weight and health surveillance recording as per IACUC guidelines. Rats were kept hydrated at all times, and the analgesic ketoprofen was administered after TBI surgery and as needed thereafter. Pre and post TBI, rats were fed with regular rodent diet from Harlan (Harlan 2018).

Post-traumatic stress disorder regimen

Rats were exposed to an adult cat for 1 hr on two occasions, separated by 10 days (Days 1 and 11). They experienced non-tactile (visual, olfactory, auditory) cues of the cat in our model of PTSD, as we described previously [16], [17], [18], [19], [20], [21]. Social instability was produced with pseudorandom changes in the pairs of cage cohorts on a daily basis. Rats experienced social instability for a total of 31 days (Days 1–31). Therefore, the two cat exposures overlapped with the period of social instability. The combination of social instability and cat exposure produces remarkable PTSD-like behavioral, physiological, endocrine and pharmacological abnormalities in stressed rats [16], [17], [18]. The TBI surgical procedure occurred one day following the second cat exposure. Thus, TBI was induced 11 days after stress induction, and then post-TBI recovery took place during a subsequent period of 20 days of stress. This approach was designed to mimic battlefield conditions in which TBI occurs in already stressed soldiers, and then their recovery must occur in conjunction with post-TBI stress.

Hematoxylin and eosin analysis

Under deep anesthesia, rats were euthanized at 8 weeks after TBI surgery, and perfused through the ascending aorta with 200 ml of ice cold phosphate buffer saline (PBS), followed by 200 ml of 4% paraformaldehyde (PFA) in PBS. H&E staining was performed to confirm the core impact injury of our TBI model. As shown in our previous studies [22], [23], [24], we demonstrated primary damage to the fronto-parietal cortex. Lesion for impacted area is approximately 29.6±9.7 mm2. In addition, H&E staining was analyzed in the hippocampus. Starting at coordinates AP-2.0 mm and ending AP-3.8 mm from bregma, coronal brain sections (40 µm) covering the dorsal hippocampus were selected. A total of 6 sections per rat was used (n = 3 randomly selected rats per group). Cells presenting with nuclear and cytoplasmic staining (H&E) were manually counted in the CA3 neurons. CA3 cell counting spanned the whole CA3 area, starting from the end of hilar neurons to the beginning of curvature of the CA2 region in both the ipsilateral and contralateral side. Sections were examined with Nikon Eclipse 600 microscope at 20X.
 
Immunohistochemistry

Under deep anesthesia, rats were sacrificed 8 weeks after TBI surgery, and perfused through the ascending aorta with 200 ml of ice cold phosphate buffer saline (PBS), followed by 200 ml of 4% paraformaldehyde (PFA) in PBS. Brains were removed and post-fixed in the same fixative for 24 hours followed by 30% sucrose in phosphate buffer (PB) for 1 week. Coronal sectioning was carried out at a thickness of 40 µm by cryostat. Staining for the cell cycle–regulating protein Ki67, migrating neuronal marker DCX, and activated microglial cell markers OX6 was done on every sixth coronal section throughout the entire striatum and dorsal hippocampus. Sixteen free-floating coronal sections (40 µm) were incubated in 0.3% hydrogen peroxide (H2O2) solution followed by 1-h of incubation in blocking solution (0.1 M phosphate-buffered saline (PBS) supplemented with 3% normal goat serum and 0.2% Triton X-100). Sections were then incubated overnight with Ki67 (1:400 Novacastra), DCX (1:150 Santa Cruz), and OX6 (major histocompatibility complex [MHC] class II; 1:750 BD) antibody markers in PBS supplemented with 3% normal goat serum and 0.1% Triton X-100. Sections were subsequently washed and biotinylated secondary antibody (1:200; Vector Laboratories, Burlingame, CA) in PBS supplemented with 3% normal goat serum, and 0.1% Triton X-100 was applied for 1 h. Next, the sections were incubated for 60 minutes in avidin–biotin substrate (ABC kit, Vector Laboratories, Burlingame, CA). All sections were then incubated for 1 minute in 3,30-diaminobenzidine (DAB) solution (Vector Laboratories). Sections were then mounted onto glass slides, dehydrated in ethanol and xylene, and cover-slipped using mounting medium.

Stereological analysis

Immunohistochemistry techniques were used in order to tag three different cell markers. Activated microglia cells were visualized by staining with OX6, an antibody against antigen presenting cell; major histocompatibility complex class ll or MHC ll+. In order to determine the cell proliferation after chronic TBI, an antibody against Ki67 (protein present in all active cell cycle phases) was used [25]. Doublecortin (DCX), an antibody against immature migrating neurons, was used to determine neuronal proliferation. Moreover, positive stainings were analyzed with a Nikon Eclipse 600 microscope and quantified using Stereo Investigator software, version 10 (MicroBrightField, Colchester, VT). The estimated volume of OX6 positive cells was examined using Cavalieri estimator probe of the unbiased stereological cell technique [26] in analyzing the cortex, striatum, thalamus, cerebral peduncle, corpus callosum, and cerebellum areas such as white matter (WM), granular cell layer (GCL), and molecular layer (ML). Ki67 and DCX positive cells were counted within the subgranular zone (SGZ) in both hemispheres (ipsilateral and contralateral), using the optical fractionator probe of unbiased stereological cell counting technique. The sampling was optimized to count at least 300 cells per animal with error coefficients less than 0.07. Each counting frame (100 X 100 µm for OX6, Ki67, and DCX) was placed at an intersection of the lines forming a virtual grid (125 X 125 µm), which was randomly generated and placed by the software within the outlined structure.

Statistical analysis

For data analyses, contralateral and ipsilateral corresponding brain areas were used as raw data providing 2 sets of data per treatment condition, therefore one-way analysis of variance (ANOVA) was used for group comparisons, followed by subsequent pairwise comparisons (post hoc tests Bonferonni test). All data are presented as mean values ± SEM. Statistical significance was set at p<0.05 for all analyses.

Results

In the preliminary analyses of the data, comparisons between sham treatment ipsilateral and sham treatment contralateral side, across all brain regions studied, did not significantly differ (p's>0.05). Thus, the data from both sides of the sham treatment groups were combined. In addition, the contralateral side across all treatment groups also did not significantly differ (p's>0.05), thus analyses were focused on comparing the ipsilateral sides from treatment groups. Analyses revealed there were no significant differences between sham and PTSD alone treatment across all brain regions examined (p's>0.05).

Upregulation of MHC ll+ activated microglia cells in chronic TBI alone and combined TBI-PTSD

To test the hypothesis of whether upregulation of microglia cells associated with chronic TBI was further exacerbated in a PTSD model with chronic TBI, different subcortical gray and WM areas were examined. The estimated volume of activated microglia cells (MHC ll+) was calculated using an anti-OX6 antibody. ANOVA revealed significant treatment effects in MHC II+ expression in the three brain regions examined (cortex, F3,20 = 11.90,***p<0.0004; striatum, F3,20 = 6.629, **p<0.0036; thalamus, F3,20 = 5.999, ** p<0.0076). Pairwise comparisons revealed TBI alone and combined TBI-PTSD resulted in a significant upregulation in the volume of MHC II-labeled activated microglia cells in gray matter areas ipsilateral to TBI when compared to PTSD alone and sham treatment (p<0.05) (Figure 1A, B, C). Moreover, TBI alone and combined TBI-PTSD, did not significantly differ between each other in the volume of MHC ll+ in ipsilateral cortex (Figure 1A), striatum (Figure 1B), and thalamus (Figure 1C) (p<0.05).

Figure 1. OX6 + expression in ipsilateral gray matter subcortical regions of TBI and TBI-PTSD rats.

Figures 1A–C represent quantitative data of estimated volumes (µm3) of OX6+ in A) cortex, B) striatum, and C) thalamus. Figures 1D–G represent OX6+ immunostaining of the ipsilateral side of cortex in D) sham-no PTSD, E) sham-PTSD, F) TBI-PTSD, G) TBI. Figures H-K represent OX6+ immunostaining of the ipsilateral side of striatum in H) sham-no PTSD, I) sham-PTSD, J) TBI-PTSD, K) TBI. Figures L-O represent OX6+ immunostaining of the ipsilateral side of thalamus in L) sham-no PTSD, M) sham-PTSD, N) TBI-PTSD, O) TBI. Note that in Figure 1C, N, the upregulation of activated microglia cells reach significance only for the group of chronic TBI combined with PTSD. Cortex, F3,20 = 11.90,***p<0.0004; striatum, F3,20 = 6.629, **p<0.0036; thalamus, F3,20 = 5.999, ** p<0.0076. Scale bars for D, E, F, G, H, I, J, K, L, M, N, O are 1 µm. doi:10.1371/journal.pone.0081585.g001

Further analysis showed significant treatment effects in MHC II+ expression in the white matter areas (corpus callosum, F3,20 = 8.611, **p<0.0017, cerebral peduncle F3,20 = 8.550, **p<0.002, fornix, F3,20 = 8.368, **p<0.002). Pairwise comparisons revealed that TBI alone and combined TBI-PTSD also instigated an increase of activated microglia cells (MHC ll+) volume in ipsilateral white matter areas compared with PTSD alone and sham treatment (p's<0.05) (Figure 2). Significant exacerbation of microglia cells in the cerebral peduncle was evident in the TBI and TBI-PTSD rats compared to PTSD alone and sham treatment (p<0.05). In addition, a significant increase in activated microglia cells in the fornix of TBI alone and TBI-PTSD group was found (p<0.05), relative to PTSD alone and sham treatment (p<0.05). TBI alone and TBI- PTSD resulted in an equivalent upregulation of activated microglia cells in corpus callosum, cerebral peduncle and fornix around the injury side (p<0.05) (Figure 2A, B, C). 

Figure 2. OX6 + expression in ipsilateral subcortical white matter regions of TBI and TBI-PTSD rats.

Figures 2 A–C represent quantitative data of estimated volumes (µm3) of OX6+ in A) corpus callosum, B) cerebral peduncle, and C) fornix. Figures 2D–G represent OX6+ immunostaining of the ipsilateral side of corpus callosum in D) sham-no PTSD, E) sham-PTSD, F) TBI-PTSD, G) TBI. Figures 2 H–K represent OX6+ immunostaining of the ipsilateral side of cerebral peduncle in H) sham-no PTSD, I) sham-PTSD, J) TBI-PTSD, K) TBI. Figures2 L-O represent OX6+ immunostaining of the ipsilateral side of fornix in L) sham-no PTSD, M) sham-PTSD, N) TBI-PTSD, O) TBI. Corpus callosum, F3,20 = 8.611, **p<0.0017, cerebral peduncle F3,20 = 8.550, **p<0.002, fornix, F3,20 = 8.368, **p<0.002. Scale bars for D, E, F, G, H, I, J, K, L, M, N, O are 1 µm. doi:10.1371/journal.pone.0081585.g002

The estimated volume of activated MHC II+ microglia cells was also calculated in the GCL, WM, and ML of the cerebellum. ANOVA revealed no detectable treatment effects on upregulation of activated microglia cells in any of the examined cerebellar regions known already the four treatment groups (Figure 3) (p's>0.05). 

Figure 3. OX6 + expression in white matter, granular cell layer and molecular layer of the cerebellum of TBI and TBI-PTSD rats.

Figure 3 A represents quantitative data of the estimated volumes (µm3) of OX6+ in three distinct regions of the cerebellum; white matter (WM), granular cell layer (GCL) and molecular layer (ML). Figures 3 B–F represent OX6+ immunostaining of the cerebellum in B) sham-no PTSD, C) sham-PTSD, D) TBI-PTSD, E) TBI. As shown in Figure 3 A, and in the photomicrographs B–E, there is no detectable upregulation of activated microglia cells in any of the examined cerebellar regions or across any of our treatment groups (sham-no PTSD, sham-PTSD, TBI-PTSD, and TBI alone); p>0.05. Scale bars for B-E are 1 µm. doi:10.1371/journal.pone.0081585.g003 

Chronic TBI impairs hippocampal cell survival and proliferation, but not neuronal differentiation in neurogenic niches

Next, we examined the effects of PTSD on chronic TBI by evaluating the total number of surviving neurons in the hippocampal CA3 region, the estimated number of positive dividing cells within SGZ, and SVZ and the estimated number of positive neuronal differentiating cells within SGZ, and SVZ where examined. ANOVA revealed significant treatment effects on neuronal survival in hippocampal CA3 (F3,8 = 13.570, **p<0.0017), with post hoc tests demonstrating that both TBI alone and combined TBI-PTSD significantly reduced CA3 cell survival in the ipsilateral hippocampus relative to ipsilateral PTSD alone and sham treatment (p's<0.05) (Figure 4A). There was no significant difference in the number of surviving neurons in the CA3 between TBI alone and TBI-PTSD animals (p>0.05) (Figure 4A). Furthermore, analyses of cell proliferation, as evidenced by number of positive Ki67 cells in the SGZ of the hippocampus, and SVZ of the lateral ventricle revealed significant treatment effects (F3,20 = 5.017, *p<0.01, F3,20 = 7.863 **p<0.0012). Post hoc tests revealed that TBI alone and combined TBI –PTSD significantly reduced cell proliferation in SGZ and SVZ in a similar manner when compared to PTSD alone and sham treatment. Both TBI alone and combined TBI-PTSD prompted a decline of proliferating cells only in the ipsilateral side of SGZ and SVZ compared to the corresponding hemispheres of PTSD alone and sham treatment (Figure 4B, and Figure 5A) (p<0.05). Finally, ANOVA revealed no significant treatment effects (F3,20 = 1.9597 ns p = 0.1512, F3,20 = 0.324 p = 0.8076) on the neuronal differentiation in the SGZ and SVZ (Figure 4C, Figure 5B). Interestingly, while cell survival and proliferation were altered by TBI and combined TBI-PTSD, there were no significant differences produced by these injuries on the neuronal differentiation across all treatment groups (p>0.05).

Figure 4. H&E, cell proliferation Ki67+, and neuronal differentiation DCX+ expressions in the hippocampus of TBI and TBI-PTSD rats.

Figures 4 A–C represent quantitative data of A) total # of neurons in hippocampal CA3, B) estimated # of Ki67+ proliferating cells in the SGZ of the DG, and C) the estimated # of DCX+ migrating cells in the SGZ of the DG. Figures 4 D–G represent H&E staining of the ipsilateral hippocampal CA3 region in D) sham-no PTSD, E) sham-PTSD, F) TBI-PTSD, G) TBI. Figures 4 H-K represent Ki67+ immunostaining of the ipsilateral SGZ of the DG in H) sham-no PTSD, I) sham-PTSD, J) TBI-PTSD, K) TBI. Figures 4 L–O represent DCX+ immunostaining of the ipsilateral SGZ of the DG in L) sham-no PTSD, M) sham-PTSD, N) TBI-PTSD, O) TBI. CA3, F3,8 = 13.570, **p<0.0017, SGZ Ki67, F3,20 = 5.017, *p<0.0106, DCX, F3,20 = 1.959 p<0.1512. Scale bars for D–G are 50 µm and H–O are 1 µm. doi:10.1371/journal.pone.0081585.g004

Figure 5. Cell proliferation Ki67+, and neuronal differentiation DCX+ expression in the SVZ of the lateral ventricle.

Figures 5 A–B represent quantitative data of A) total # of Ki67+ cells representing the number of cell proliferating in the SVZ, B) the estimated # of DCX+ migrating cells in the SVZ of the lateral ventricle. Figures 5 C–F represent Ki67+ immunostaining of the ipsilateral SVZ in C) sham-no PTSD, D) sham-PTSD, E) TBI, F) TBI-PTSD. Figures 5 G–J represent DCX+ immunostaining of the ipsilateral SVZ of the lateral ventricle in G) sham-no PTSD, H) sham-PTSD, I) TBI, J) TBI-PTSD. SVZ Ki67, F3,20 = 7.863, **p<0.0012, SVZ DCX, F3,20 = 0.324, ns p = 0.8076. Scale bars for C–J are 50 µm. doi:10.1371/journal.pone.0081585.g005

Discussion

In the present in vivo study, we demonstrated that exacerbation of activated microglia cells was detected at 8 weeks in chronic TBI and is associated with CA3 cell loss, and dysfunctional cell proliferation in the hippocampus. In this first assessment of histological pathology, we have found that PTSD did not exacerbate TBI-induced neuroinflammation and neurodegeneration. After eight weeks post-TBI, chronic TBI alone, and chronic TBI combined with PTSD showed a similar significant augmentation of intensified activated microglia cells in cortical and subcortical regions.

There was a 20-fold increase of activated microglia cells in cortex for TBI alone and TBI combined with PTSD when compared to PTSD alone and sham treatment. There was a 12-fold increase of activated microglia cells in both TBI and TBI-PTSD in the striatum. The TBI-PTSD group showed 30-fold increase relative to sham treatment; however, it failed to reach statistical significance when compared with TBI alone group. For the TBI alone and TBI-PTSD, there were 10-fold and 30-fold increments, respectively, of activated microglia cells present in the thalamus. The corpus callosum showed 100-fold increases of active microglia cells compared with PTSD alone and sham treatment. In the cerebral peduncle area, there were 10-fold and 15-fold increments in TBI and TBI-PTSD, respectively, relative to PTSD alone and sham treatment. The area of the hippocampal fornix showed 10-fold and 8-fold increments in TBI and TBI-PTSD, respectively, compared with PTSD alone and sham treatment. Furthermore, cerebellar analysis showed that there were no significant increments of activated microglia cells in the white matter (WM), granular cell layer (GCL), and molecular layer (ML) relative to sham treatment. In parallel, distinct hippocampal areas were assessed to determine neurodegeneration. Both TBI and TBI-PTSD showed significant declines of CA3 hippocampal neurons relative to stress and sham treatment. By comparing our TBI model with the TBI-PTSD model, we can conclude that chronic PTSD exposure did not further generate an increase in neuronal cell death in the CA3 region of the hippocampus. Of note, analysis of the contralateral side showed no significant differences in CA3 cell loss across treatment groups.

Further examination of the hippocampus revealed significant declines in the proliferative capacity of newly born cells within the SGZ. There was about 40% decrease in cell proliferation within the SGZ in both TBI and TBI-PTSD when compared with PTSD alone and sham treatment. The analysis showed that the injury caused by TBI alone was associated with impaired cell differentiation in the SGZ of the hippocampus since there was no further impairment on cell proliferation when TBI is combined with PTSD. Moreover, histological analysis of the SVZ showed that the proliferative ability of the cells in this neurogenic niche was also affected by chronic TBI alone which was not further impaired when TBI was combined with PTSD. Of note, in both neurogenic niches, SGZ and SVZ, the cell differentiation profile was not affected by either TBI alone or by the combination of TBI and PTSD.

To our knowledge, experiments using animal models of TBI exposed to PTSD to evaluate synergistic effects in neuroinflammation and neuronal degeneration are currently lacking, representing a major gap of knowledge on the interaction between TBI and psychological morbidities, such as PTSD. There is one study that assessed the influence of chronic stress on blast-related traumatic brain injury in rats [27]. As in the current work, these investigators documented brain injury in response to stress and TBI. However, as they did not include a non-stress TBI group, it cannot be confirmed that stress exacerbated the histopathology they observed. Thus, the issue of the conditions, brain areas and aspects of histopathology affected by stress-TBI interactions remains unresolved. This lack of conclusive scientific evidence for stress-TBI interactions also highlights the complexity behind the pathological mechanisms that link chronic head traumas and PTSD in TBI survivors. Nonetheless, while the present observations do not show PTSD exacerbation of TBI-induced histopathological deficits, these findings provide compelling results that can be used to guide future studies on the biological bases of TBI and its association with the development of neuropsychological morbidities post-TBI or the worsening of pre-existing conditions. For example, the current work focused on assessment of histological pathology in response to PTSD and brain trauma. It will be important in subsequent work to include an analysis of how stress-TBI interactions are expressed at behavioral and cognitive levels, in conjunction with histopathological assessments.

Long-term consequences of chronic TBI may be related to increased risk for chronic neuroinflammation, neurodegenerative diseases, executive function impairments, and the development of neuropsychological disorders such as anxiety, dementia, depression and PTSD [5], [6], [13], [23], [28], [29], [30], [31], [32], [33]. A growing body of literature suggests that TBI and PTSD are becoming the signature morbidities of our military personnel and veterans [5], [6], [25]. Clinical studies show that TBI is significantly associated with limited functional impairments, while TBI comorbid with PTSD and depression was significantly associated with chronic long lasting cognitive deficits in servicemen following deployment [5], [6]. In addition, animal studies show that exposure to blast injuries induced psychological abnormalities and increments of proteins that enhance fear responses for several months after the initial exposure [32]. In the clinic, patients with a history of brain injury display neuropsychological disturbances affecting executive function, attention and memory [34], which may be mediated by reduced cerebral blood flow in the thalamus, a brain structure implicated in neurological impairments such as memory and learning and verbal respond speed [35]. These findings are in agreement with the present results which depict a significant exacerbation of activated microglia cells in dorsal thalamic nuclei in animals exposed to TBI combined with PTSD.

The present results also showed that TBI and TBI with PTSD produced an equal extent of hippocampal neurodegeneration. However, PTSD alone did not alter the hippocampal CA3 survival. Similarly, TBI and combined TBI- PTSD, but not PTSD alone, reduced the cellular proliferative capacity in both neurogenic niches of SGZ and SVZ, while neuronal differentiation was not significantly impaired in any of the treatment groups. These results support previous clinical studies, showing a lack of decrease in hippocampal volume in TBI-PTSD patients [15], [36], [37], [38], [39], [40], [41], [42], [43]. Our findings are in agreement with these clinical studies, whereby chronic stress did not exacerbate the neuropathological effects of TBI alone. However, these findings warrant further investigations in view of previous work demonstrating that stress, particularly elevated levels of corticosterone, can exacerbate damage to the hippocampus in response to metabolic insults, including hypoxia or administration of neurotoxins [44]. Thus, whether the reported stress-induced exacerbation of brain damage from metabolic challenges can be distinguished from potential stress-TBI interactions remains to be determined.

Finally, clinical studies have found that there is a close association between patients that suffer TBI-and a traumatic psychological event, with decreased hippocampal neuronal volume. In addition, these studies showed that these patients have an increased risk for the development of more severe neuropsychological disorders such as severe depression, bipolar disorder and PTSD [15]. There is also evidence suggesting that there is a strong link between TBI and worsening of psychological conditions such as depression and PTSD[15], [45], [46], [47], [48], [49], [50], [51], [52], [53]. To this end, whether hippocampal cell loss and alteration in neurogenic capacity are associated with the development of TBI co-morbidities is not clear. Further studies addressing the biological bases on how TBI combined with neuropsychological stress and more severe PTSD impairs hippocampal function, such as long-term potentiation and memory formation, are needed.

Conclusions

To our knowledge, this is the first laboratory report of histopathological characterization of TBI-PTSD. This study reveals that the combined TBI-PTSD group displayed similar neuroinflammation and impaired cell proliferation profiles. Other TBI-mediated cell death events, such as oxidative stress and apoptosis, warrant further investigations. Investigations of TBI and its co-morbidity factors will allow a better understanding of the disease pathology and guide treatment that will address both primary and secondary cell death events, as well as psychological and physical impairments.

Author Contributions

Conceived and designed the experiments: CVB DMD PRS. Performed the experiments: SAA SW NT KS HI DGH CVB. Analyzed the data: SAA NT CVB. Contributed reagents/materials/analysis tools: DMD PRS YK CVB. Wrote the paper: SAA DMD CVB. Designed psychosocial stress paradigm: DD. Contributed analysis: PRS YK.

References available at the PLoS ONE site

Jerome Wakefield - Psychiatric Diagnoses: Science or Pseudoscience?

 

This is an interesting podcast from the good folks at the Institute for Ethics and Evolving Technologies (IEET) on the validity of psychiatric diagnoses - a very relevant topic here at the Evolution of Psychotherapy Conference. Many of the speakers we have heard so far do not use the DSM diagnostic protocols because they have little to do with the clients we see in our offices.



Jerome Wakefield is the author of The Loss of Sadness: How Psychiatry Transformed Normal Sorrow into Depressive Disorder (2007), All We Have to Fear: Psychiatry's Transformation of Natural Anxieties into Mental Disorders (2012), and several other books.

Psychiatric Diagnoses: Science or Pseudoscience?

Rationally Speaking
Posted: Dec 10, 2013 


Jerome Wakefield, DSW, PhD

The standard for diagnosis is the Diagnostic and Statistical Manual of Mental Disorders (DSM), which just released a 5th edition in 2013—but just how objective is it? This episode of Rationally Speaking features Dr. Jerome Wakefield, psychiatrist, PhD in philosophy, and author of "The Loss of Sadness: How Psychiatry Transformed Normal Sorrow into Depressive Disorder." Julia, Massimo and Jerome talk about the arbitrariness of the DSM and the controversies around the boundaries of various mental disorders, including depression and sexual fetishes.



Jerome's pick: Bertran Russells's Autobiography

Listen/View

New research from Richard Davidson's team at the U of Wisconsin has identified gene expression changes in meditation. After eight hours of mindfulness practice, the meditators showed a range of genetic and molecular differences, including altered levels of gene-regulating machinery and reduced levels of pro-inflammatory genes, which in turn correlated with faster physical recovery from a stressful situation.

The full article is available online (the link is below the summary). Here is a summary of the findings followed by the abstract from the full article.

Gene Expression Changes With Meditation

Dec. 8, 2013 — With evidence growing that meditation can have beneficial health effects, scientists have sought to understand how these practices physically affect the body.

A new study by researchers in Wisconsin, Spain, and France reports the first evidence of specific molecular changes in the body following a period of mindfulness meditation.

The study investigated the effects of a day of intensive mindfulness practice in a group of experienced meditators, compared to a group of untrained control subjects who engaged in quiet non-meditative activities. After eight hours of mindfulness practice, the meditators showed a range of genetic and molecular differences, including altered levels of gene-regulating machinery and reduced levels of pro-inflammatory genes, which in turn correlated with faster physical recovery from a stressful situation.

"To the best of our knowledge, this is the first paper that shows rapid alterations in gene expression within subjects associated with mindfulness meditation practice," says study author Richard J. Davidson, founder of the Center for Investigating Healthy Minds and the William James and Vilas Professor of Psychology and Psychiatry at the University of Wisconsin-Madison.

"Most interestingly, the changes were observed in genes that are the current targets of anti-inflammatory and analgesic drugs," says Perla Kaliman, first author of the article and a researcher at the Institute of Biomedical Research of Barcelona, Spain (IIBB-CSIC-IDIBAPS), where the molecular analyses were conducted.

The study was published in the journal Psychoneuroendocrinology.

Mindfulness-based trainings have shown beneficial effects on inflammatory disorders in prior clinical studies and are endorsed by the American Heart Association as a preventative intervention. The new results provide a possible biological mechanism for therapeutic effects.

The results show a down-regulation of genes that have been implicated in inflammation. The affected genes include the pro-inflammatory genes RIPK2 and COX2 as well as several histone deacetylase (HDAC) genes, which regulate the activity of other genes epigenetically by removing a type of chemical tag. What's more, the extent to which some of those genes were downregulated was associated with faster cortisol recovery to a social stress test involving an impromptu speech and tasks requiring mental calculations performed in front of an audience and video camera.

Perhaps surprisingly, the researchers say, there was no difference in the tested genes between the two groups of people at the start of the study. The observed effects were seen only in the meditators following mindfulness practice. In addition, several other DNA-modifying genes showed no differences between groups, suggesting that the mindfulness practice specifically affected certain regulatory pathways.

However, it is important to note that the study was not designed to distinguish any effects of long-term meditation training from those of a single day of practice. Instead, the key result is that meditators experienced genetic changes following mindfulness practice that were not seen in the non-meditating group after other quiet activities -- an outcome providing proof of principle that mindfulness practice can lead to epigenetic alterations of the genome.

Previous studies in rodents and in people have shown dynamic epigenetic responses to physical stimuli such as stress, diet, or exercise within just a few hours.

"Our genes are quite dynamic in their expression and these results suggest that the calmness of our mind can actually have a potential influence on their expression," Davidson says.

"The regulation of HDACs and inflammatory pathways may represent some of the mechanisms underlying the therapeutic potential of mindfulness-based interventions," Kaliman says. "Our findings set the foundation for future studies to further assess meditation strategies for the treatment of chronic inflammatory conditions."

Study funding came from National Center for Complementary and Alternative Medicine (grant number P01-AT004952) and grants from the Fetzer Institute, the John Templeton Foundation, and an anonymous donor to Davidson. The study was conducted at the Center for Investigating Healthy Minds at the UW-Madison Waisman Center.

* * * * *

Rapid changes in histone deacetylases and inflammatory gene expression in expert meditators

Perla Kaliman, Marıa Jesus Alvarez-Lopez, Marta Cosın-Tomas, Melissa A. Rosenkranz, Antoine Lutz, Richard J. Davidson

Full Citation:
Kaliman, P, Alvarez-Lopez, MJ, Cosın-Tomas, M, Rosenkranz, MA, Lutz, A, Davidson, RJ. (2014). Rapid changes in histone deacetylases and inflammatory gene expression in expert meditators. Psychoneuroendocrinology; 40, 96—107. DOI: 10.1016/j.psyneuen.2013.11.004

Summary

Background: A growing body of research shows that mindfulness meditation can alter neural, behavioral and biochemical processes. However, the mechanisms responsible for such clinically relevant effects remain elusive.

Methods: Here we explored the impact of a day of intensive practice of mindfulness meditation in experienced subjects (n = 19) on the expression of circadian, chromatin modulatory and inflammatory genes in peripheral blood mononuclear cells (PBMC). In parallel, we analyzed a control group of subjects with no meditation experience who engaged in leisure activities in the same environment (n = 21). PBMC from all participants were obtained before (t1) and after (t2) the intervention (t2 t1 = 8 h) and gene expression was analyzed using custom pathway focused quantitative-real time PCR assays. Both groups were also presented with the Trier Social Stress Test (TSST).

Results: Core clock gene expression at baseline (t1) was similar between groups and their rhythmicity was not influenced in meditators by the intensive day of practice. Similarly, we found that all the epigenetic regulatory enzymes and inflammatory genes analyzed exhibited similar basal expression levels in the two groups. In contrast, after the brief intervention we detected reduced expression of histone deacetylase genes (HDAC 2, 3 and 9), alterations in global modification of histones (H4ac; H3K4me3) and decreased expression of pro-inflammatory genes (RIPK2 and COX2) in meditators compared with controls. We found that the expression of RIPK2 and HDAC2 genes was associated with a faster cortisol recovery to the TSST in both groups.

Conclusions: The regulation of HDACs and inflammatory pathways may represent some of the mechanisms underlying the therapeutic potential of mindfulness-based interventions. Our findings set the foundation for future studies to further assess meditation strategies for the treatment of chronic inflammatory conditions.

Charles Eisenstein - The More Beautiful World Our Hearts Know Is Possible (Free Webinar)

Here the free video of a recent webinar given by Charles Eisenstein on his new book, The More Beautiful World Our Hearts Know Is Possible - on sacred activism. Good stuff.

Charles Eisenstein - The More Beautiful World Our Hearts Know Is Possible

Published on Dec 10, 2013


Author of Sacred Economics: Money, Gift, and Society in the Age of Transition (2011), The Ascent of Humanity: Civilization and the Human Sense of Self (2007), and The More Beautiful World Our Hearts Know Is Possible (Sacred Activism) (2013), Charles Eisenstein discusses ideas from his newest book, The More Beautiful World Our Hearts Know Is Possible, during a free webinar on December 10, 2013 before embarking on the second leg of his world tour in January.

• Discover how small, individual acts of courage, kindness, and self-trust can change our culture's guiding narrative of separation.
• Learn how to overcome cynicism, frustration, and paralysis in the face of our current planetary crisis.
• Become a more effective agent of positive change through increased awareness of interconnectedness and interbeing.

Presented by http://NABCommunities.com/

Katy Davis - The Power of Vulnerability (Animated Video from The RSA)

Big thanks to Brene Brown for posting this on her blog - it's a sweet little video.

This Gives New Meaning to Bear Hug! An RSA Short Animated by Katy Davis

December 10, 2013

So grateful to The RSA (Royal Society for the encouragement of Arts) for inviting me to speak in London this year and to animator and illustrator, Katy Davis, for this amazing short on empathy! Beautiful.

Evolution of Psychotherapy Conference - My Schedule

I am at the Evolution of Psychotherapy Conference in Anaheim, CA, just up the street from Disney Land (the happiest place on Earth, or something like that).

There is so much to do and learn - and some of it overlaps. Some of the biggest names in psychotherapy over the last 30 years are speaking here, including Otto Kernberg, Albert Bandura, Gerald Edelman (1972 Nobel Prize in Physiology/Medicine), Donald Meichenbaum, Judith Beck (daughter of Aaron Beck), Herriet Lerner, Ernest Rossi, Cloe Mandanes, Irvin Yalom, and Bessel van der Kolk, as well as some younger stars, such as Mary, Pipher, Francine Shapiro, Daniel Siegel, Stephen Gilligan, Peter Levine, and Steven Hayes.

Anyway, here is my schedule for the conference.

WEDNESDAY

8:30-9:30 - Keynote 1: Anaheim Convention Center

GERALD EDELMAN, MD, PHD - FROM BRAIN DYNAMICS TO CONSCIOUSNESS:  How Matter Becomes Imagination Arena

10:00-1:00 - DANIEL SIEGEL, MD - MINDFULNESS, MINDSIGHT AND THE BRAIN: What is Mind and Mental Health? - Hilton - Pacific Ballroom

2:30-5:30 – OTTO KERNBERG - TRANSFERENCE FOCUSED PSYCHOTHERAPY (TFP) OF SEVERE PERSONALITY DISORDERS - Marriott - Platinum 6-10

THURSDAY

8:30-11:30 – JEFFREY ZEIG, PHD:  HYPNOSIS: Advanced Techniques for Beginners - Marriott - Platinum Ballroom 1-5

1:00-4:00 – STEPHEN GILLIGAN, PHD - THE THREE POSITIVE CONNECTIONS: NEEDED FOR CREATIVE CHANGE - Marriott - Platinum Ballroom 6-10

4:30-5:30 – DANIEL AMEN, MD - BRAIN WARS: How Not Looking at the Brain Leads to Missed Diagnoses, Failed Treatments and Dangerous Behaviors - Hilton - California Ballroom

5:45-6:45 – MICHAEL GAZZANIGA, PHD - UNITY IN A MODULAR WORLD - Anaheim Convention Center - Arena

FRIDAY

8:00-9:00 – Daniel Siegel, MD - The Wheel of Awareness and the Integration of Consciousness (Live) - Anaheim Convention Center - Arena

9:20-10:20 – Robert Dilts - Accessing and Applying Archetypal Energies as Resources for Change and Healing (Live) - Hilton - California Ballroom

10:40-11:40 – PANEL: Posttraumatic Disorders - Jack Kornfield, PhD, Peter Levine, PhD, Donald Meichenbaum, PhD, and Mary Pipher, PhD, Moderator: Annellen Simpkins, PhD - Hilton Pacific Ballroom

12:00-1:00 – BESSEL VAN DER KOLK, MD - FRONTIER OF TRAUMA TREATMENT - MODERATOR: ANNELLEN SIMPKINS, PHD - Hilton - California Ballroom

2:30-3:30 – Stephen Gilligan, PhD - Generative Trance and Transformation (Live) -

Anaheim Convention Center Arena

3:50-4:50 – PANEL: Transference/Countertransference Otto Kernberg, MD, Peter Levine, PhD, and Erving Polster, PhD, Moderator: Michael Munion, MA - Marriot Grand Ballroom

5:10-6:10 – Ernest Rossi, PhD - The Mind-Body Healing Experience (MHE) (Live) - Anaheim Convention Center Arena

SATURDAY

8:00-9:00 – Steven Hayes, PhD - Compassion and Perspective Taking in Acceptance and Commitment Therapy (Video) - Marriott Grand Ballroom

9:20-10:20 – Donald Meichenbaum, PhD - Treatment of a Suicidal Patient with a History of Victimization: A Constructive Narrative Perspective (Video) - Hilton - Pacific Ballroom

10:40-11:40 – ALBERT BANDURA, PHD - ON SHAPING ONE’S FUTURE - MODERATOR: ALEXANDER SIMPKINS, PHD - Anaheim Convention Center Arena

12:00-1:00 – PETER LEVINE, PHD - SPIRITUALITY AND TRAUMA, MODERATOR: ALEXANDER SIMPKINS, PHD - Anaheim Convention Center Ballroom ABC

2:30-5:30 – BESSEL VAN DER KOLK, MD - THE BODY KEEPS SCORE: Integration of Mind, Brain, and Body in the Treatment of Trauma - Hilton California Ballroom

7:00-9:00 – IRVIN YALOM, MD - TEACHING PSYCHOTHERAPY THROUGH NARRATIVE - Anaheim Convention Center Arena

SUNDAY

8:00-9:00 – Steven Hayes, PhD - SELF, COMPASSION, AND PEACE OF MIND: The Implications of Evolution Science, Moderator: Betty Alice Erickson, MS - Marriott - Platinum Ballroom 6-10

9:15-11:15 – MICHAEL D. YAPKO, PHD - REALITY IS NEGOTIABLE: Absorbing People in Positive Possibilities - Marriott - Platinum Ballroom 1-5

11:30-12:30 – AARON T. BECK—COGNITIVE THERAPY PAST, PRESENT AND FUTURE PATHWAYS: Cognitive Therapy Past, Present and Future Pathways: A DISCUSSION WITH CHRISTINE PADESKY, PHD - AARON BECK, MD INTERVIEWED BY JUDITH BECK, PHD - Anaheim Convention Center Arena

12:30-1:30 - MARTIN SELIGMAN, PHD - POSITIVE PSYCHOLOGY: NEW DEVELOPMENTS - Anaheim Convention Center Arena