Nerve Questions
What is the normal resting potential in a nerve?
-60 to -70mV
What are the sizes of sensory neurons?
0.2-15um
What are the sizes of motor neruons?
3-20um
What size of defect are autografts limited to?
5cm in length (Daly 2012). beyond that requires allograft
Define peripheral nerves (Slide 3)
All of the nerves outside the CNS, which includes the brain and spinal cord
What is the success rate of Autografts?
Daly 2012 states that the success rate is 50% of patients. (may be due to the fact that sensory nerves, ex. sural, are being used to repair motor and mixed nerves. This causes morphometric mismatches in the natvie environments- mismatch in axonal size, distribution and alignment)
Who is said to have done the first nerve tubulation experiments?
Gluck in 1881. He said that the experiments failed due to scar formation.
why are biodegradable conduits perferred over non-biodegradable ones?
In general, biodegradable materials are preferred because nonbiodegradable ones eventually may lead to compression. (ruiter 2009)
What is the speed of conduction down a nerve?
Normally 50-60m/s; however the range can be from 0.2 to 120 m/s depending on the individual and the nerve type
What are the different cell types in the CNS and what do they do?
Oligodendrocytes (myelinate CNS axons; provide structural framework) Astrocytes (maintain blood-brain barrier; provide structural support; regulate ion, nutrient, and dissolved gas concentrations; absorb and recycle neurotransmitters; form scar tissue after injury) Microglia (remove cell debris, wastes, and pathogens by phagocytosis) Ependymal cells (line ventricles(brain) and central canal (spinal cord); assist in producing circulating, and monitoring of cerebrospinal fluid)
why is tensionless repair needed?
Prior studies have shown that injury tends to occur when nerves are stretched to greater than 10% of their original length. It may even initiate the process with stretching as little as 4%-6%[3,4]. Negative outcomes have been reported with tension greater than 25 g[5]. (Gerth 2015) Tension has been found to hamper regeneration, possibly by promoting scar formation and adhesions (1,2). Tension also impairs blood supply to the nerves (3,4). In particular, Flores et al (3) found that an induced 8% elongation causes a 46% decrease in perfusion, thereby potentially limiting regeneration of transected peripheral nerves. (Mcdonald 2010) An article by Sunderland et al (9) provided evidence that nerves can withstand a certain degree of stretching before negatively affecting regeneration. In their rat sciatic nerve model, gaps of up to 6 mm could be closed primarily with subsequent anatomical and functional recovery, similar to that obtained with a gap of 0 mm. Only when the nerves were stretched to overcome a 9 mm gap (corresponding to approximately 0.56 N of tensile force) was there a demonstrable negative impact on regeneration. Sunderland et al (9) hypothesized the existence of a 'tension threshold' and proposed that nerves can indeed be stretched without detriment up to an intrinsically determined point. (Mcdonald 2010)
What are the different cell types in the PNS and what do they do?
Satellite cells (surround neuron cell bodies in ganglia; regulate O2, CO2, nutrient, and neurotransmitter levels around neurons in ganglia) Schwann Cells (surround axons in PNS; are responsible for myelination of peripheral axons; participate in repair process after injury)
What happened in 1882?
The first successful application of a hollow NGC where a hollow bone tube was used to bridge a 30mm nerve gap in a dog. Done by Vanlair.
What is Neurorrhaphy?
The joining together, usually by suture, of the two parts of a divided nerve
Why are current hollow NGC limited in size?
This limitation is thought to be due to the inadequate formation of ECM components during the initial stages thus leading to a lack of guidance cues and a reduction of the formation of the bands of Bungner; and causing the regenerating fibers to stop short and form stumps rather than bridging the gap
45. What are some issues?
a. "conduts have failed over increased nerve diameters, a failure primarily caused by incorrect NGC conduit design [6] (Daly 2012) b. "The first generation of artificial nerve conduits used in the clinic were nonresorbable silicone tubes, which were plagued by compression syndrome and often required secondary surgeries for removal (13)" (Nectow 2012) c. The current generation of conduits provides mainly physical guidance cues via conduit morphology to help direct damaged nerve to its target sensory and motor outputs. The next generation of conduits will seek to augment nervous system function by using topographical and protein cues that interact with nervous tissue at the cellular level" (Nectow 2012)
17. What is that a figure of (slide 3)
a. 32 y/o male; he had had an injury to the median nerve from stab wound. Median nerve exploration was performed and a neuroma was identified. The neuroma was resected and the interfascicular graft (sural nerve cables were used) was placed.
What are the types of nerves and what are there function?
a. A alpha: Motor-skeletal muscle b. A beta: Sensory-touch, pressure c. A gamma: motor-musle spindles; proprioception d. A delta: fast pain; temperature e. B: Autonomic; Pre-ganglionic; sympathetic f. C (unmyelinated): slow pain, autonomic, postganglionic sympathetic, polymodal nociceptors
19. What is a fascicle and what is the difference between a fascicle and the Perineurium? (slide 4)
a. A fascicle is a bundle or a cluster' (there are both muscle and nerve fascicles in the body) b. Basically, the perineurium is the 'covering' of the fascicle, which is a bundle of nerve fibers
What is the difference between bed disability days and restricted activity?
a. All disability days (and therefore, bed disability days) are considered restricted activity days i. Restricted activity-when a person cuts down on his usual activities for the whole of that day on account of an illness or an injury ii. Bed disability days-when a person stays in bed for all or most of the day
13. What is meant by market? (slide 3)
a. An area or arena in which commercial dealings are conducted
52. What is Embed 812
a. An embedding resin i. Use resin to preserve the morphology of the tissue
38. Why use a rat at 200g?
a. At that point, the rat is about 7 to 8 weeks old. This is associated with sexual maturity (which actually occurs at 1.5 months). This corresponds to late teens in humans. The systems are all balanced
40. What are the FDA classes?
a. Class I: Class I devices are deemed to be low risk and are therefore subject to the least regulatory controls. For example, dental floss is classified as Class I device. b. Class II: Class II devices are higher risk devices than Class I and require greater regulatory controls to provide reasonable assurance of the device's safety and effectiveness. For example, condoms are classified as Class II devices c. Class III: Class III devices are generally the highest risk devices and are therefore subject to the highest level of regulatory control. Class III devices must typically be approved by FDA before they are marketed. For example, replacement heart valves are classified as Class III devices.
39. What FDA class will this device be?
a. Class II
33. What are the current allograft options
a. Currently there is only one FDA approved accellular nerve allograft (the Avance graft from AxoGen) b. 1885-first nerve allograft transplantation was reported.
51. Why do you dehydrate tissue samples for histology?
a. Dehydration is simply the removal of water from aqueous-fixed tissue. Since most fixatives are aqueous, this step is necessary to prepare the tissue for embedding in non-aqueous media like paraffin. Alcohols are most commonly used in the laboratory for tissue dehydration, since they are miscible with aqueous fixatives like 10% formalin. In this step, the alcohol penetrates tissue quickly and the water is replaced with alcohol. Since alcohols act rapidly and may shrink and harden tissue too much, care must be taken when calculating the amount of time needed in the dehydration step. This step is performed at room temperature. Ethyl alcohol and isopropyl alcohol are used most often, with methanol and butanol being used to some degree in special techniques. Since most alcohols (with the exception of isopropyl alcohol) and paraffin are NOT miscible, another step, known as clearing, is introduced.
20. What is the importance between the layers of connective tissue? (Slide 4)
a. Dense irregular: collagen fibres are arranged in bundles w/o a definitie orientation. This provides resistance to stress from all directions. This type of tissue is encountered in the dermis. The epinerium is usually most abundant around joints, as its function is to protect nerves from stretching and subsequent injury i. Few fibroblasts and some macrophages; thick collagen ii. Epineurium: b. Dense regular: collagen bundles are arranged according to a definite pattern). Tendons are the most common example of dense regular connective tissue. i. Only fibroblasts; parallel collagen and some elastic ii. Perineurium: c. Loose: the most common type; it holds organs in place and attaches epithelial tissue to other underlying tissues. It also surrounds blood vessels and nerves; fibroblasts are widely dispersed in this tissue; primarily made up of collagenous and elastic fibers i. Endoneurium: consists mainly of collagen fibrils; some elastic and reticular (type III collagen) (mostly made by SCs)
What is the speed related to?
a. Diameter of the nerve and degree of myelination b. Low temperature slows down nerve conduction c. Age. NCV is low in infants and children (in neonates, it is nearly half of the adult values. It attains the adult value by three to five years of age. Then remains relatively stable until 60y/o; after which it starts declining at a rate of 1.5 % per decade)
22. What is myelin? (slide 4)
a. Fatty white substance; outgrowth of either SCs or oligodendrocytes; insulator b. "The production of the myelin sheath is called myelination. In humans, myelination begins in the 14th week of fetal development, although little myelin exists in the brain at the time of birth. During infancy, myelination occurs quickly, leading to a child's fast development, including crawling and walking in the first year. Myelination continues through the adolescent stage of life c. Myelin was discovered in 1854 by Rudolf Virchow d. Myelinated axons are white in appearance, hence the 'white matter' of the brain. e. Cholesterol is an essential constituent of myelin. Myelin is about 40% water
47. What is the main ingredient of the band of bungner?
a. Fibronectin
53. What does 1% toluidine blue do/
a. Has an affinity for nucleic acids b. Collagen-pale blue c. Cytoplasm-pale blue d. Nuclei-dark blue e. Myelin-dark blue to black
26. So the 3rd and 4th degree NEVER regenerate on their own?????? (slide 4)
a. I would hesitate to say 'never' because the body continues to amaze us; however, the common train of thought is that these types of injuries require outside help for full recovery
32. How are non-critically sized defects fixed?
a. If possible, nerve endings are sutured together
35. What are the problems with current NGC?
a. In a peer reviewed published clinical study of empty nerve tubes, 100% meaningful recovery was reported in gaps of less than 5 mm1 however other studies found 34% of repairs failed for gaps of 5 mm or greater1 , 35% of repairs had tube related complications3 and 31% of repairs required revision in gaps 2.5 - 20 mm (sensory, mixed, motor)
42. How is Collagen I formed?
a. Inside the cell i. Two types of alpha chains are formed during translation on ribosomes along the rough endoplasmic reticulum(RER): alpha-1 and alpha-2 chains. These peptide chains (known as preprocollagen) have registration peptides on each end and a signal peptide. ii. Polypeptide chains are released into the lumen of the RER. iii. Signal peptides are cleaved inside the RER and the chains are now known as pro-alpha chains. iv. Hydroxylation of lysine and proline amino acids occurs inside the lumen. This process is dependent on ascorbic acid (vitamin C) as a cofactor. v. Glycosylation of specific hydroxylysine residues occurs. vi. Triple alpha helical structure is formed inside the endoplasmic reticulum from two alpha-1 chains and one alpha-2 chain. vii. Procollagen is shipped to the Golgi apparatus, where it is packaged and secreted by exocytosis. b. Outside the cell i. Registration peptides are cleaved and tropocollagen is formed by procollagen peptidase. ii. Multiple tropocollagen molecules form collagen fibrils, via covalent cross-linking (aldol reaction) by lysyl oxidase which links hydroxylysine and lysine residues. Multiple collagen fibrils form into collagen fibers. iii. Collagen may be attached to cell membranes via several types of protein, including fibronectin and integrin.
30. What does the word 'Chromatolysis' mean and how is this important in nerve regeneration? (slide 6)
a. It is the dissolution of the Nissl bodies in the cell body of a neuron. The event of chromatolysis is also characterized by a prominent migration of the nucleus towards the periphery of the cell and an increase in the size of the nucleolus, nucleus, and cell body b. The regeneration and repair phase following nerve injury may last for many months. The earliest signs of this phase are visible changes in the cell body that mark the reversal of chromatolysis. The nucleus returns to the cell center and nucleoproteins reorganize into the compact Nissl granules. Postinjury, many subcellular metabolic functions were altered during chromatolysis. Likewise, RNA synthesis was increased and neurotransmitter synthesis decreased. Chromatolysis heralded a fundamental shift in cell function from synaptic transmission to cellular repair. The metabolic machinery was reprogrammed so that the cell would be able to produce the vast amount of protein and lipid needed for axonal regrowth during the regeneration phase
14. Who did the market study? (slide 3)
a. Magellen medical Technology Consultants, Inc, Minneapolix, MN
43. Who was the first to do protein stamping
a. Microcontact printing was developed by the whitesides group (at Harvard) in the mid-1990s. This specific protocol that we will use in this endeavor was published by the Fienberg group at Carnegie Mellon University.
48. Schwann Cells
a. Most studies focus on adding schwann cells to the surface of the nerve tissue engineering scaffolds (DING 2011) b. Schwann cells line up along external lamina to form the band of Bunger (DING 2011) (Arslantunali 2014) c. Schwann cells provide the necessary adhesion surface to induce and guide growth cone migration (DING 2011) d. Schwann cells stimulate and regulate axonal regeneration and myelin formation (DING 2011) e. Schwann cells facilitate axon maturation and nerve reinnervation (DING 2011) f. Secrete ECM such as laminin and fibronectin (DING 2011) g. Current problems with using schwann cells i. Immune rejections and ethical concerns of heterologous schwann cells (DING 2011) ii. for autologous schwann cells 1. 2 operations needed, increasing suffering (DING 2011) 2. Problem of how to generate a large number of schwann cells in vitro and maintain high purity (DING 2011) 3. Problem of decreased secretion of neurotrophic factors in the graft (DING 2011) 4. Need to enhance the ability of transplanted Schwann cells to migrate (DING 2011) 5. Schwann cells lack the directional guidance needed for regeneration of axons (17,18 of DING 2011)
27. What are the first cells on site in an injury in the body? Is this the same for nerve injuries and is it vital? (slide 6)
a. Other tissues: neutrophils(leukocytes; scavenger cells that remove damaged tissue and infectious agents; bystander damage to healthy cells is a byproduct of neutrophil activation and release of microbicidal products) are the first cells to respond to injury; macrophages clean up debris; mast cells induce swelling, warmth and redness; all 3 types of cells summon more immune system cells b. Systemic treatment of mice with an anti-Ly6G antibody to deplete neutrophils, cells that play an essential role in the genesis of neuropathic pain, did not affect recovery of neurological function and peripheral axon regeneration
41. What are the 2 main amino acids found in collagen? Glycine (non-polar; neutral) and proline (non-polar; hydrophobic)Glycine is found at almost every third residue.
a. Proline makes up about 17% of collagen.
44. When were NGC's first used?
a. Proposed for use for nerve repair as early as 1881 with the first successful application occurring in 1882, where a hollow bone tube was used to bridge a 30mm nerve gap in a dog
21. What are SCs and what is their function? (slide 4)
a. SCs are the principal glia (non-neuronal cells that maintain homeostasis, form myelin, and provide support and protection for neurons in the CNS and PNS) of the PNS b. 2-types: myelinating and nonmyelinating i. Myelinating SCs wrap around axons of motor and sensory neurons to form the myelin sheath (1 axon per cell; covers about 100um of an axon)—this decreases the membrane capacitance. Myelinating SCs begin to form the myelin sheath in mammals during fetal development and work by spiraling around the axon, sometimes with as many as 100 revolutions. A well-developed SC is shaped like a rolled-up sheet of paper, with layers of myelin in between each coil. ii. Nonmyelinating SCs hold axons in invaginations. These are involved in maintenance of axons and are crucial for neuronal survival.
31. What donor nerves are usually used?
a. Sensory nerves (ex. Sural nerve, superficial radial sensory, and medial antebrachial cutaneous nerve)
25. What does the 3rd and 4th sunderland degrees correspond with in the Seddon classification system? (slide 4)
a. Since the sunderland classification was made secondarily, it is filling in gaps missing from the seddon classification. Though axonotmesis (class II) was initially described as an injury to just the myelin sheath and axon; 3rd and 4th degree sunderland injuries are associated as being a class II injury.
29. How fast can a nerve regenerate (slide 6)
a. Small (2mm/day); large (5mm/day)
36. Why did you choose an n=6?
a. The ASTM (American society for testing and materials) standard for biological materials is a minimum of 6. Later we can use this data to do a power analysis for future studies
23. What is the band of bungner? (slide 4)
a. The SC columns are known as the bands of Bungner and become important guides for sprouting axons during reinnervation
What are the main divisions and subdivisions of the PNS and what do they do?
a. The autonomic NS: Regulates involuntary bodily processes, including heart rate, respiration, digestion and pupil contraction; operates-automatically without conscious direction (motor innervation of smooth muscle, cardiac muscle, and glands) i. Sympathetic NS: Prepares the body for action and stress ii. Parasympathetic NS: calms the body and helps conserve energy b. The somatic NS: Carries sensory information from sensory organs to the CNS and relays motor(movement) commands to muscles; controls voluntary movements; (motor innervation of all skeletal muscles)
15. Define quality of life (QOL) (slide 3)
a. The degree to which a person enjoys the important possibilities of his or her life" (UofT)
50. Why is osmium tetroxide used in the staining procedure?
a. The most important fixation reactions of osmium tetroxide are those involving unsaturated bonds of lipids and phospholipids as it is one of the few fixatives that stabilises lipids. b. It can also be used as an en bloc stain for demonstrating lipids (particularly myelinated nerve fibres) at the light microscope level.
12. What group of trauma patients is that statement referencing and how did they define trauma? (Slide 3)
a. The study references a trauma population of 5,777 patients treated between Jan 1, 1986 and Nov 30, 1996 at sunnybrook health science centre (a regional level 1 trauma center)
28. What is the time frame of regeneration (in a more detailed scale)? (slide 6)
a. Wallerian degeneration (anterograde degenerative changes): within 24hrs, neurofilaments break up and the axon fragments into short lengths. Within 10 days, the myelin sheath breaks down into lipid droplets around the axon. Within 30days the myelin is denatured chemically. Within 3 months, macrophages from the endoneurium invade the degenerating myelin sheath and axis cylinder. The macrophages phagocytose the debris, leaving behind only the endoneural tube b. Retrograde degenerative changes ('affect the dendritic tree, the cell body, and the part of the axon proximal to the lesion". i. Acute response: starts after 24hrs-48hrs (and peaks in about 2 wks) and is characterized by the disintegration of Nissl substance into fine dust (represents inactive, aggregated RNA). 'following chromatolysis, the RNA disperses in the cytoplasm, ennabling the neuron to mobilize the protein synthesis required for regeneration. ii. Chronic response: follows the acute reponse only occasionally. It involves the atrophy of the nerve cell body with all its processes." c. Nerve rengeneration repair 'begins about 20days after nerve section and is complete in 3 months. Regenerative changes usually occur simultaneously in the cell and the axon." i. Axon: up to 100 axonal sprouts, each containing a neurofibril, grow out in all directions from the proximal axon BUT only 1 will survive. 'the schwann cells in the Band of Bungner form myelin sheath around the reinnervating axonal sprout. The sheath begins to develop in about 15 days and is complete within a year. Increase in fiber diameter takes place very slowly. Regenerated fibers attain a fiber diameter of up to 80 percent of normal. ii. Cell body: 'the nissl substance and golgi apparatus gradually reappear. Sometimes, they reappear in greater than the original amounts, reflecting the extra metabolic effort required to replenish large amounts of the axoplasm. The cell regains its normal size and the nucleus returns to its central position. d. (wiki) macrophages and SCs release neurotrophic factors that enhance re-growth('most neurotrophic factors belong to 1 of 3 families: 1)neurotrophins; 2)glial cell-line derived neurotrophic factor family ligands (GFLs), and 3)neuropoietic cytokines. Each family has its own distinct signaling family though the cellular responses elicited often do overlap.
Do the CNS and PNS have different healing capabilities?
a. Yes, CNS regeneration is much slower, and is almost absent in most vertebrate species. The primary cause for this could be the delay in clearing up myelin debris (which contains several inhibitory factors). i. Schwann cells (PNS) can survive w/o axon signals and they help remove debris as well as call in macrophages to help. ii. However, Oligodendrocytes (CNS) require axon signals to survive—with axon death, they either die or enter a state of rest. They don't clean or ask for help (no macrophage recruitment). Furthermore, Macrophage entry in general into CNS site of injury is very slow. (In contrast to PNS, Microglia play a vital role in CNS wallerian degeneration---but microglia recruitment is still slow....~3days; plus they aren't reliable(might not become fully phagocytic cells)...plus they are slow at cleaning compared to macrophages) b. There is a delay in Wallerian degeneration in CNS compared to PNS—possibly due to the difference in clearance rates of myelin in CNS and PNS
16. Define morbidity (slide 3)
a. a diseased state, disability, or poor health due to any cause
18. Tell us about the median nerve (slide 3)
a. originates from the brachial plexus; has contribution from ventral roots C5, C6, and C7 (all 3 from the lateral cord) and C8&T1(from the medial cord). The median nerve is the only one that passes through the carpal tunnel. i. What type of nerve is it? Mixed (afferent-sensory; efferent-muscle) ii. What does it do? Innervates the (all the flexors except flexor carpi ulnaris and part of flexor digitorum profundus) anterior compartment of the forearm (with two exceptions), thenar eminence, lumbricals, skin of the hand
46. How long is immune suppression required for allografts?
a. requires the use of extensive immune suppression up to 18 months post implantation, and patients become susceptible to opportunistic infections, occasionally resulting in tumour formation [18] (Daly 2012)
24. Which system was made first? (slide 4)
a. seddon (1943); sunderland (1951)
NCV-Nerve conduction velocity (AKA. NCS-nerve conduction study)
i. How is it performed? (concept: apply shock at one point of the nerve and record the signal from another) ii. Analyzing the test results. If slower than normal->sign of damage to the myelin sheath. Decreased response but normal speed -> damage to the nerve fiber
EMG-Electromyogram
i. How is it performed? Needle electrode is inserted into a muscle; electrical activity is recorded for rest and contraction ii. Analyzing the test results/causes for abnormal results. 'electrical activity in a muscle at rest (should be none) shows that there may be a problem with the nerve supply to the muscle. Abnormal wave lines when a muscle contracts may mean a muscle or nerve problem
define anastomosis
medically: "a connection made surgically between adjacent blood vessels, parts of the intestine, or other channels of the body, or the operation in which this is constructed."
why are swelling and degradation important nerve tube properties?
swelling might occlude the lumen for regeneration or cause compression of regenerated axons. The rate of degradation might contribute to this swelling by the formation of small degradation products that increase the osmotic pressure of the conduit.10,14 Too rapid degradation may lead to swelling, but too slow degradation may later lead to compression and/or a chronic foreign body reaction. The ideal nerve tube should remain intact for the time axons need to regenerate across the nerve gap and then degrade gradually with minimal swelling and foreign body reaction. As for the mechanical properties, by changing the nerve tube dimension14 or copolymer ratio,10 the swelling and degradation properties may be optimized
define coaptation
the drawing together of the separated tissue in a wound or fracture.