Biophotonics - Traumatic Brain injury and Neuroimaging | BIM 289A, Study notes of Biology

Download Biophotonics - Traumatic Brain injury and Neuroimaging | BIM 289A and more Study notes Biology in PDF only on Docsity! Selected Topics In Biophotonics: Traumatic Brain Injury and Neuroimaging Dr. Gene Gurkoff, Ph.D. UCD Department of Neurological Surgery and Center of Biophotonics S&T Outline • My Background • Neuroscience and Biomedical Engineering • Traumatic Brain Injury • Applying Neurophotonics to Trauma Neuroscience and BME: A Real Terminator? Fully implantable multichannel recording device Wireless link Portable Sy controller F Mechanical actuators with both power and accuracy Touch and position sensors Remote Control Rats? 3 Talwar et al 2002 How Might You Ask? • Implanted Stimulating Electrodes into the Median Forebrain Bundle • Additional Electrodes in the Right and Left Somatosensory Cortex Whisker Representations. • Mounted a Backpack Containing a Microprocessor-based, Remote controlled Microstimulator • Using a Laptop, Rats Could be Guided From Distances up to 500 Meters Away Talwar et al 2002 Diseases of Neuroscience: Genetic: Huntington’s Parkinson’s Alzheimer’s FragileX Spinocerebellar Ataxia Triplet Repeat: Huntington’s FragileX Spinocerebellar Ataxia Degenerative: Huntington’s Parkinson’s Alzheimer’s Stroke Epilepsy TBI Cognitive: Huntington’s Alzheimer’s Stroke Epilepsy TBI Fragile X Behavioral: Huntington’s Parkinson’s Stroke Epilepsy TBI Pain Developmental: FragileX Spinocerebellar Ataxia Epilepsy “Environmental”: Parkinson’s Alzheimer’s Stoke Epilepsy TBI Chronic Pain Specific Targets? • Genetic Disorders – Gene Therapy – Protein Replacement/Removal • Environmental Disorders – Reduce Exposure • Less pesticides • Better Diet • Exercise • Protection Stroke, Epilepsy and TBI • These are more diffuse brain injuries affecting multiple cell types in multiple locations in a variety of ways. • These can happen at different stages of life and can have different consequences in the very young, young, adult and elderly. • No Early Signs • Is there a Therapeutic Window? Yes! Annual Incidence of TBI 50,000 Deaths 235,000 Hospitalizations 1,111,000 Emergency Department Visits ??? Receiving Other Medical Care or No Care At least 1.4 million reported TBIs occur in the United States each year.* * Average annual numbers, 1995-2001 based on a study from the CDC Other Diseases? • Alzheimer’s: 377,000 in 1995 (Herbert et al 2001). Average age at onset is 72.1 years (Li et al 2002). • Parkinson’s: 1.5 million people (total) live with and approximately 60,000 new cases are diagnosed each year (Annual Conference of Mayors 2003). Average age of onset is 60.1 years (Li et al 2002). • Cardiovascular disease accounts for 38% (1.4 million) of all deaths in 2002 (CDC). • Autism: A total of 118,602 (Department of Education). This number is rising as awareness increases. • 475,00 children (0-14)suffer a TBI each year • TBI is the #1 cause of death and disability in children. • The second largest group of individuals receiving TBI are in the 15-19 year old age group - people your age. Head Trauma Bad!! Helmets Good!! • Only known cure for head trauma is to not get a head trauma – Wear your Helmets!! – Wear your Seatbelts!! My Specific Hypothesis • My interest has always been TBI in the younger population - especially as related to multiple insults • Majority of standard of care related to repeat injury to date is anecdotal. • It has always been assumed that two injuries in close temporal proximity will result in more anatomical damage and increased behavioral deficits over an extended period of time. Is this the case? Specific Aim 1: To determine the window of vulnerability post-TBI where seizures will induce an increase in cell death as assessed 24-hours post-injury compared to either injury alone. Pilocarpine or Saline (s.c.) 1, 6 or 24 hrsP19 Severe LFP or Sham Perfusion 24 Hours H&E/Cell Counts Paraffin Controls: Sham LFP alone LiPc alone Experimental Groups: LFP + LiPc P18 Pretreatment 3mEq LiCl (s.c.) LFP Does Not Protect Against LiPc- Induced Epileptogenesis 20-Weeks Post- Injury Does Pilocarpine Treatment Lead to Epilepsy? 0% 25% 50% 75% Controls (n=32) LFP (n=14) LiPc (n=15) LFP+LiPc (n=15) % O f E p il e p ti c A n im a ls Seizure Threshold Test: Pentylene- Tetrazol (PTZ) • Weighed the rat • Infused 10 mg/ml PTZ through the tail vein at 3 mL/hr • Measured time to 1st forelimb myoclonus and generalized seizure 0 5 10 15 20 25 30 35 40 45 Forelimb Myoclonus Generalized Seizure M e a n P T Z C o n c e n tr a ti o n ( m g / k g ) ± S E M No Epilepsy Epilepsy* * Epilepsy and PTZ-induced Seizures LFP Does not reduce mossy fiber sprouting? 0 0.5 1 1.5 2 Control (N=12) LFP (N=8) LiPc (N=7) LFP+LiPc (N=7) A v e r a g e T I M M S c o r e ± S E M * * • 0 - No Granules Between Crest and Tips of Supragranular Layer • 1 - Occasional Granules in the Supragranular Region Occurring in Patchy Distribution • 2 - Numerous Granules in the Supragranular Region Occurring in Patchy Distribution • 3 - Granules in the Supragranule Region Occurring in Near Continuous Distribution • Assessed 3 Caudal Sections of Dorsal Hippocampus (200 µm interval) Summary Of Data • Our hypothesis was that we would see vulnerability. There was no statistical vulnerability in any measure • Acutely TBI seemed to protect neurons from subsequent seizure-induced death • This preconditioning did not have an effect on long--term outcome measures Conclusions of Study • Interesting from a clinical standpoint for several reasons – Suggests that there may be a therapeutic window post-TBI – What is TBI doing that protects the brain? Can we promote this physiological response? – Saving cells acutely does not necessarily lead to a better outcome • Patients care about their behavior, not how many cells they lost. Neurons MOTOR NEURON MITRAL CELL FROM PYRAMIDAL CELL FROM SPINAL CORD OLFACTORY BULB FROM CORTEX Uf able Dendrite —_ — _— Dendrite _— Dendrite Axon — PURKINJE CELL How Do Neurons Communicate? The Action Potential +40 Membrane potential (mV) -70 Na+ channels al become refractory, no more Nat enters cell K+ continues to leave cell, causes membrane K* channels + potential to return open, K ‘ to resting level begins to leave 9 cell Na+ channels open, Nat begins to enter cell Ww K+ channels close, Na* channels reset Se Threshold of Extra K* outside excitation diffuses away K+ Na+ K+ Na+, Ca2+ K+ Na+, Ca2+ Axon Dendrite Axon Dendrite K+ Na+ K+ Na+ X 10 K+ Na+ X 10 Mechanism Responsible for Acute Preconditioning to Cell Death • There is an acute elevation of extracellular potassium following LFP in the adult rat. • Slices taken following LFP in the developing rat identified elevated potassium concentrations in the hippocampus up to 48 hours post-injury Katayama et al, 1991 J Neurosurg D’Ambrosio et al, 1999 J Neurosci How Do Neurons Communicate? The Action Potential +40 Membrane potential (mV) -70 Na+ channels al become refractory, no more Nat enters cell K+ continues to F leave cell, causes membrane K* channels + potential to return open, K to resting level 22? a annels close, channels reset Se Threshold of Extra K* outside excitation diffuses away begins to leave cell Na+ channels open, Nat begins to enter cell a K+ Na+, Ca2+ K+ Na+, Ca2+ K+ Na+ X ? K+ Na+ X 5 Axon Dendrite Axon Dendrite K+K+ K+ Na+ X 5 K+ Na+ X 10 ? Design Modified Ringers Glutamate 100uM or 1mM or Mild or Moderate Stretch Injury Stop Recording 10 min Record Δ in Na+, Ca2+, Lactate and pH Load Cells 10 min 30 min Modified Ringers Glutamate 100uM or 1mM or Mild or Moderate Stretch Injury Quantify Cell Death 10 min 24 hours Groups: Ringers K+[3] K+[10] K+[30] K+[60] K+[90] Preliminary Data Normalized % Cell Death + SEM 120 5 100 - 80 4 60 4 40 4 20 4 0 K[10] K[30] K[65] K[90] fetal (N=10) (N=10) (N=12) (N=10) Conclusions • Preliminary data seems to suggest that manipulating the extracellular concentration of potassium can protect cells from excitotoxic insult. • Can it do the same for mechanical insult? • Where do I go from here? More Advanced Microscopy • Spinning Disk Confocal Microscopy – Advantages: • High resolution • Full field • Fast (better than video rate) • Great for biological reactions – Disadvantages: • Photobleaching and phototoxic • More expensive, more technical CCD camera Filter Wheel (emissions) Filter Wheel (excitation) Dichromatic Mirror Confocal Disk Mirror Cube Objective Lamp Specimen Using SERS to Look at pH • Epi-fluoresence – Advantages: • Fast (biological reactions) • Full Field • Affordable – Disadvantages: • Lower resolution • High background • Photobleaching and photoxic • Surface Enhanced Raman Spectroscopy (SERS) – Advantages: • High resolution • Full field (with spinning disk) • Great for biological reactions • No photobleaching – Disadvantages: • Slower • Higher Variability • Not full coverage of a cell • How do we get them into a cell? Injury Perfusion (Day 1) Cryoprotect (Day 2) Freeze/Cut (Day 4) Stain/Mount (Day 6) Coverslip (Day 7) Count (Day 8) Interval Dynamic Changes Post-Injury • Blood Brain Barrier Study: – 1 injury severity – 3 different [pharmaceutical] + controls – 5 time points (45, 90 and 180 min, 24 and 72 hrs) • 1 severities X 6 groups X 8 animals per group X 5 time points = 240 animals • 1-hour to count each region of interest • 2 regions bilaterally = 4 hours/brain X 240 animals = 960 hours of quantification (96 10-hour days) What does one want from microscopy?• Speed • Resolution • Large field of view • Tissue penetration • In-vivo and in-vitro • Non-Invasive • No probes or good, easy to use probes • No photoxicity/bleaching • The reality is that there is no holy grail of microscopy (yet). There are advanced systems that do address multiple needs • Collaboration across disciplines is increasing demand to really push the limits of neuroscience My Personal Experience (Playing hooky on Thursday February 7th, 2008)