ILP z3186431

From CellBiology

An Investigation of the Pathophysiology of Delirium

--Mark Hill 11:03, 16 May 2009 (EST) Project background information added to your discussion page.

Summary: Pathophysiology of Delirium- A Focus on Apoptosis

Delirium and Dementia

The pathophysiology of delirium itself remains a little understood concept1, and little research has been conducted on apoptosis and delirium specifically. However, research on dementia is more extensive. There is a connection between these conditions (similar clinical pictures, outcomes, hypotheses and a relatable link between having one condition and getting the other); it may be possible that research into dementia can be applied to delirium.

Apoptosis

Apoptosis is the predominant form of cell-death in chronic neurodegenerative diseases2. These diseases include Dementia, Parkinson’s, and Huntington’s.

Summary of the Process of Apoptosis

Several apoptotic pathways exist, mainly the intrinsic and extrinsic pathways which can then be subdivided. In both pathways, signalling results in the activation of caspases3. Caspases are proteases (cysteine-dependent, aspartate-specific proteases) that are the major executioners in the apoptotic program 2,3,4. Caspases have been categorized into upstream initiators and downstream executioners. Upstream caspases are activated by the cell-death signal. These upstream caspases activate downstream caspases, which directly mediate the events leading the cell’s demise2.

Downstream, or executioner, caspases (caspase-3, -6, -7) are thought to be responsible for the direct destruction of the cell5. The contribution that each executioner caspase makes to the execution phase of apoptosis remains speculative, with especially caspase-6 and -7’s role remaining unclear 5. The different pathways will now be briefly looked at.

Extrinsic Pathways

The Extrinsic pathway begins outside a cell, when conditions in the extracellular environment determine that a cell must die6. The extrinsic pathway of apoptosis is activated by cross-linking members of the tumor necrosis factor (TNF) receptor superfamily, such as Fas and death receptors DR4 and DR5, by their receptors, Fas ligand or TRAIL (TNF-related apoptosis-inducing ligand), respectively7. This induces the association of Fas-associated death domain (FADD) and procaspase-8 to death domain motifs of the receptors. Caspase-8 is activated and then cleaves and activates effector caspases-3 and -7, which then target cellular constituents inducing apoptosis6.

Intrinsic Pathways

The major mechanism of death in the intrinsic pathway is via mitochondria. However, more recent research has indicated other pathways may play a role including those modulated by the Endoplasmic Reticulum (ER) and the nucleus.

Mitochondrial Pathway

The major trigger of apoptosis in the mitochondrial pathway is Cytochrome-C2, 3. In the cytoplasm, cytochrome-c binds to Apaf-1(for Apoptosis Protease-Activating Factor2,3). The result of this binding is called an apoptosome — a molecular complex consisting of cytochromec , Apaf-1, ATP, and procaspase-92,3. The apoptosome activates caspase-9, an upstream initiator of apoptosis. Cytochrome-C is regulated by the Bcl-2 family2,3. There are pro-apoptotic (e.g. Bax, Bad, Bid) and anti-apoptotic (e.g. Bcl-2, Bcl-XL) members of this family4. The balance between proapoptotic and antiapoptotic signals from the Bcl-2 family has a crucial role in the release of cytochrome-c2. This balance may be affected in neurodegenerative diseases, and could be an avenue for further research in neurodegenerative diseases and delirium specifically.


Markers of cell death

There are markers that exist that reflect neuronal destruction8. Examples of these include Neuron-specific enolase (NSE), a glycolytic enzyme that is located in neuronal cytoplasm, and S100B, a calcium-binding protein localized to astroglial cells9. It has been proposed that after brain damage these neuronal and/or glial proteins are released from brain cells. Increased CSF levels of proteins such as NSE, S-100B, MBP and GFAP have been observed in acute neurological disorders such as cerebrovascular accidents, traumatic brain injury, exacerbating multiple sclerosis (MS) and in chronic progressive disorders such as Creutzfeldt–Jacob Disease (CJD) and dementia 8.9. Delirium itself has been linked with decreased levels of NSE in CSF and plasma (Personal communication, Caplan). It is unknown why the levels of NSE decreased instead of increased; one theory is that the decline in NSE may indicate a disruption to the glycolytic pathway (Personal communication, Caplan). Lactate dehydrogenase (LDH) is another accepted marker for cellular damage. LDH is released by all types of cells after loss of membrane integrity10. CSF-LDH activity is increased in central nervous system (CNS) infections, head trauma, vascular accidents, and metastatic CNS disease27

Glial fibrillary acidic protein (GFAp) is an astrocyte-specific protein11. This is important as the cell death present in delirium may indeed not be only of the neurons but in the cells that support them, the astrocytes. GFAp was determined in cerebrospinal fluid (CSF) of adults with global cognitive dysfunction11. GFAp values in CSF were increased in 65 of 121 samples of adults with dementia, independent of its cause11. This is another avenue future delirium research can go down.


References

1 Mukadam N, Ritchie CW, Sampson EL. Cholinesterase Inhibitors for delirium: what is the evidence? International Psychogeriatrics. 2008;20(2):209-18

2Friedlander RM. Mechanisms of Disease: Apoptosis and Caspases in Neurodegenerative Diseases. New England Journal of Medicine. 2003;348:1365-75. 3 Takuma K, Yan SS, Stern DM, Yamada K. Current Perspective: Mitochondrial Dysfunction, Endoplasmic Reticulum Stress, and Apoptosis in Alzheimer’s Disease. J Pharmacol Sci 2005: 97, 312 – 316

4 Kermer P, Liman J, Weishaupt JH, Bähr M. Neuronal Apoptosis in Neurodegenerative Diseases: From Basic Research to Clinical Application Key Players in Neuronal Apoptosis. Neurodegenerative Dis 2004;1:9–19

5 Slee EA, Adrain C, Martin SJ. Executioner Caspase-3,-6 and -7 perform distinct non-redundant roles during the demolition phase of apoptpsis. J Biol Chem. 2001:276(10):7320-6.

6 Kumar V, Abbas A, Fausto N. Robbins and Cotran Pathological Basis of Disease. 7 e.d. USA: Elsevier Saunders 1999.

7 Fauci SA, Braunwald E, Kasper DL, Hauser SL, Longo DL, Jameson JL, Loscalzo J. Harrison's Principles of Internal Medicine 17 e.d. USA: The McGraw-Hill Companies. 2009. Accessed online from http://www.accessmedicine.com/resourceTOC.aspx?resourceID=4

8 H. Schmidta, M. Ottoa, P. Niedmannb, L. Cepeka, A. Schröterc, H.A. Kretzschmard, S. Posera CSF Lactate Dehydrogenase Activity in Patients with Creutzfeldt-Jakob Disease Exceeds That in Other Dementias Vol. 17, No. 3, 2004 dementia and geriatric cognitive disorders.

9 Berger RP, Pierce MC, Wisniewski SR, Adelson PD, Clark RS, Ruppel RA, Kochanek PM. Neuron-specific enolase and S100B in cerebrospinal fluid after severe traumatic brain injury in infants and children. Pediatrics. 2002;109(2):E31

10 Herrmann M, Ebert AD, Galazky I, Wunderlich MT, Kunz WS, Huth C. Neurobehavioral outcome prediction after cardiac surgery: role of neurobiochemical markers of damage to neuronal and glial brain tissue. Stroke. 2000;31(3):645-50

11 R. Crols R, Saerens J, Noppe M, Lowenthal A. Increased GFAp levels in CSF as a marker of organicity in patients with Alzheimer's disease and other types of irreversible chronic organic brain syndrome. Journal of Neurology 1986; 233(3): 157-160


Comparison of Elisa Kits on Aoptosis

Each kit will be duscussed in terms iof Type, Markers, Suppliers, Sensitivity, Cost, Overseas/domestic, Usage, in that order:


1) Sandwich ELISA Kit #7190 Cleaved Caspase-3 (Asp175) PathScan®  - TBA O/S 1


2) Human Active Caspase-3 Quantikine ELISA Kit KM300 Caspase 3 R&D Systems 0.1 ng/mL TBA O/S 1


3) Human Caspase-3 BMS2012INST Caspase 3 Bender MedSystems 0.12 ng/ml Assay Range: 10 - 0.16 ng/ml TBA O/S 1

 

4) ELISA Kit Catalog # KHO1091 Caspase 3 Invitrogen 0.033 ng/mL $828 Domestic Supplier 1


5) Apoptosis Multi-Target Sandwich ELISA Kit #7105 p53 protein, phospho-p53 protein (Ser15), Bad, phospho-Bad (Ser112), Cleaved Caspase-3 (Asp175) and Cleaved PARP (Asp214)* PathScan - TBA O/S 1


6) RD 192072200R Human GFAP ELISA Gfap BioVendor Analytical Limit of Detection is calculated from the real GFAP values in wells and is 0.033ng/ml Assay Sensitivity takes the dilution of samples into consideration and is calculated according to the formula: Assay Sensitivity = Analytical Limit of Detection x sample dilution = 0.033ng/ml x 3 =0.1ng/ml TBA O/S 1


7) Sandwich enzyme immunoassay KA0024 Human GFAP (Glial Fibrillary Acidic Protein) Abnova Corporation - $985 O/S 1


8) Bcl-2 Sandwich ELISA Kit APT230 Bcl-2 Family Chemicon International 3.9 ng/mL Range of detection: 250 ng/mL to 3.9 ng/mL Detects human Bcl-2 protein in cell lysates, supernatants, whole blood, and serum. TBA Domestic supplier Australia +61 3 9839 2000 1


9) Human Cytochrome c EIA Kit 900-141 Cytochrome C Assay Designs/Stressgen Bioreagents 6.03 pg/ml Assay range: 28.13-900 pg/ml TBA Domestic Supplier 1


10) Human Cytochrome c EIA Kit 650.070.096 Cytochrome C Diaclone 0.05 ng/ml TBA  1


11) Human Cytochrome c EIA Kit RBMS263R Cytochrome C. BioVendor Laboratory Medicine, Inc. 0.05 ng/ml TBA  1


12) Immunoassay Kit Catalog #KHO1051 Cytochrome C Invitrogen <0.156 ng/mL $829 Domestic Supplier 1


13) Human CD178 / FasL ELISA Kit 850.750.096 Fas-L Diaclone/Tepnel 44pg/ml Range: 62.5-2000 $350 Domestic supplier 1


14) Human CD178 / FasL ELISA Kit 850.750.192 Fas-L Diaclone/Tepnel 44pg/ml Range: 62.5-2000 $525  2  



1)      http://www.cellsignal.com/products/7190.html


2)      www.rndsystems.com/pdf/km300.pdf


3)       http://www.biocompare.com/RequestInformation/8/892214/Human-Caspase-3-Instant-ELISA-Kit-from-Bender-MedSystems.html?q=1


4)      http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&productID=KHO1091


5) http://www.cellsignal.com/products/7105.html


6) www.ibl-america.com/pdf/BioVender/RD192072200R_IBL.pdf


7) http://www.abnova.com/Products/Products_Detail.asp?strCountry=US&Catalog_ID=KA0024


8) http://www.millipore.com/searchsummary.do?tabValue=&q=bcl-2+elisa


9) http://www.assaydesigns.com/commerce/ccp1155-1155-cytochrome-c-28human29--eia-kit.htm


10) http://www.tepnel.co.uk/product-details.asp?ID=552&Cat=397


11) http://www.biovendor.com/country315/product/immunoassays/cytochrome-c-human-elisa


12) http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&entryPoint=adirect&productID=KHO1051&messageType=catProductDetail


13) http://www.diaclone-usa.com/diaclone/datasheet.asp?A=kits&S=850.750.192


14) http://www.diaclone-usa.com/diaclone/datasheet.asp?A=kits&S=850.750.192

Final Decision on ELISA kits to be Used

1) Type: Human VEGF ELISA Kit

Code: ELH-VEGF-001

Supplier:RayBio

Sensitivity: 20pg/mL

Domestic supplier: Yes. Order on thursdays.

Cost:$653

Gst: $65.30

Shipping: FREE

Contact: Bioscientific Pty Ltd.

Tel: 1-300-246-724, 9521-2177

Fax: 61 (0)2-9542 3100

Web: http://www.biosci.com.au

Address: 28 Monro Ave Kirrawee, NSW 2232, Australia

Email: info@biosci.com.au


2) Type: Human Bcl-2 ELISA Kit

Code: BMS244/3 & BMS244/3TEN

Supplier:Bender medsystems

Sensitivity: 0.5ng/mL

Domestic supplier: Yes. They order fortnighlty. next order can be placed by TUESDAY 21ST OF JULY. It takes 2 weeks to arrive (cannot order again until the 11th of AUGUST)

Cost:$1,097

Gst: $109.70

Shipping: $44

Contact: http://www.bendermedsystems.com/bm_products/MAN/244-3TEN.pdf or JOMAR BIOSCIENCE Pty. Ltd. P.O.Box 385, Unit 2, 15 Maesbury Street Kensington SA 5068

Tel.: +61 8 8431 2041 Fax: +61 8 8431 2049 jomar@adelaide.on.net www.jomar.on.net


3) Type: Human GFAP ELISA

Code: KA0024

Supplier: Abnova

Sensitivity: 0.1ng/ml

O/S or Domestic: Local distributor:

Sapphire Bioscience Pty Ltd.

Ms. Melissa Gliddon (Customer Service)

Ph: +61 2 9698 2022

Fax: +61 2 9698 1022

e-mail: service@sapphirebioscience.com

Cost:$1436 Au INCLUDING GST

Shipping: $20

NB: Ordering takes place every M0NDAY and WEDNESDAY and will take 2-3 weeks to arrive


4)type: fas-l

Abcam , with Sapphire Bioscience Pty Ltd as distributor Telephone: +61 2 9698 2022

Specificity Range: 62.5 pg/ml - 2000 pg/ml

Sensitivity < 44 pg/ml

price: $812 excluding gst, delivery cost $20. midday on tues and thurs. 2-3 wks delivery

http://www.abcam.com/Human-Fas-Ligand-ELISA-Kit-1-x-96-Well-Plate-ab45892.html

Cytochrome C ELISA Practice Experiment

Aim:

To determine if Cytochrome C is detectable in the serum and CSF of patients with persistent delirium, when compared against dementia controls.

Method:


The kit used was Chemicon International Cytochrome C Elisa Kit (Cat No. APT200, LOT NUMBER: VR1351309

The procedure were followed according to the intructions. A list of materials used is below:


Materials:

1. Anti-Cytochrome C antibody coated plate (Part no. 90155): 96 wells

2. Biotinylated Anti-cytochrome c monoclonal antibody (part no. 90157): one 110 uL vial

3. Steptavidin-Enzyme conjugate (Part no: 90158): one 50 uL vial

4. Cytochrome c standard (part no: 90156): one lyophilized vial

5. Assay dilutent (part no 90159): 30mL bottle

6. Wash buffer (part no. 60245): 100mL bottle

7. Substrate solution )part no. 60096): 0ne 10ml bottle

8. Stop solution (part no. 60193): one 12ml bottle

9. Refrozen thawed vials of CSF and Blood from 35 patients


Preparing the plate:

After leaving two blank wells, a standard curve was then generated by making serial dilutions in the order of 1:2, starting backwards from 40 ng/ml, then continuing 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml, 0.675ng/ml, 0.337ng/ml, 0.168ng/ml, 0.084ng/ml

50 uL aliquots of serum and CSF from 35 patients were tested. These were placed in duplicate on the plate.


Reading the plate

Following the steps of the Elisa, the plate was read by a plate reader, Multiskan FC (thermoscientific) at an absorbance 450nm.

The prinicipal of the test dictates that a coloured product is formed in proportion to the amount of Cytochrome C present.

A standard curve is prepared from the standard dilutions, and sample cytochrome C concentration is then determined.


Results:

The average absorbance for each duplicated group was used.

This average was plugged into an equation formulated by the standard curve

The results were negative for all but one, so could not be appropriately plotted on the curve. (See excel graph)


Conclusion:

No result able to be demonstrated.

Negative numbers may indicate that the cytochrome C was not within the detection range.


Discussion:

There are several possible reasons why no result was yielded from this experiment.

1. The blanks were not blank

2. The duplicates did not demonstrate the same results, when they should have been close to identical

3. The plate reader was not sufficiently programmed in time for the completion of the ELISA. due to the time sitting atround, a colour change may have occurred. This is in accordance with the instruction manual which states results should be read immediately, as colour will fade over time

Areas for improvement include more precise pipetting so that the correct volume is found in the wells, especially for duplicates of the same sample.

As well as this, the successful completion of a standard curve is what the entire experiment hinges on. The concentrations of sample cytochrome C is dependent on the stansdard being accurate, so the fact that the standard curve in this experiement had negative nuimbers fior its raw data conteributed to the experiments failure.

Preparedness before the time of the experiment is crucial, for example if the plate reader had been properly set up beforehand, the experiment would have run more smoothly. As well as this the serial dilutiions could also have been calculated beforehand.

Instructions for BCL-2 elisa kit

Preparation of Reagents

Buffer concentrates should be brought to room temperature and should be diluted before starting the test procedure.

9.1 Wash Buffer If crystals have formed in the Wash Buffer Concentrate (20x), warm it gently until they have completely dissolved. Pour entire contents (50 ml) of the Wash Buffer Concentrate (20x) into a clean 1000 ml graduated cylinder. Bring to final volume of 1000 ml with glass-distilled or deionized water. Mix gently to avoid foaming. The pH of the final solution should adjust to 7.4. Transfer to a clean wash bottle and store at 2° to 25°C. Please note that Wash Buffer is stable for 30 days.

9.2 Assay Buffer (1x) Pour the entire contents (5 ml) of the Assay Buffer Concentrate (20x) into a clean 100 ml graduated cylinder. Bring to final volume of 100 ml with distilled water. Mix gently to avoid foaming. Store at 2° to 8°C. Please note that the Assay Buffer (1x) is stable for 30 days.

9.3 Lysis Buffer Pour the entire contents (15 ml) of the Lysis Buffer Concentrate (10x) into a clean 150 ml graduated cylinder. Bring to final volume of 150 ml with distilled or deionized water and mix gently. Store at room temperature. Please note that the Lysis Buffer is stable for 30 days.

9.4 Biotin-Conjugate Please note that the Biotin-Conjugate should be used within 30 minutes after dilution. Make a 1:100 dilution of the concentrated Biotin-Conjugate solution with Assay Buffer (1x) in a clean plastic tube

9.5 Streptavidin-HRP Please note that the Streptavidin-HRP should be used within 30 minutes after dilution. Make a 1:100 dilution of the concentrated Streptavidin-HRP solution with Assay Buffer (1x) in a clean plastic tube as needed

9.6 Human Bcl-2 Standard Reconstitute human Bcl-2 standard by addition of distilled water. Reconstitution volume is stated on the label of the standard vial. Swirl or mix gently to insure complete and homogeneous solubilization (concentration of reconstituted standard = 64 ng/ml). After usage remaining standard cannot be stored and has to be discarded. Standard dilutions can be prepared directly on the microwell plate (see 10.d) or alternatively in tubes (see 9.6.1). 9.6.1 External Standard Dilution Label 7 tubes, one for each standard point. S1, S2, S3, S4, S5, S6, S7 Then prepare 1:2 serial dilutions for the standard curve as follows: Pipette 225 μl of Sample Diluent into each tube. Pipette 225 μl of reconstituted standard (concentration = 64 ng/ml) into the first tube, labelled S1, and mix (concentration of standard 1 = 32 ng/ml).

Pipette 225 μl of this dilution into the second tube, labelled S2, and mix thoroughly before the next transfer. Repeat serial dilutions 5 more times thus creating the points of the standard curve (see Figure 6). Sample Diluent serves as blank.

Test Protocol a. For cell lysis follow the cell lysate protocol (see 6.1 Sample Preparation – Cell Lysate Protocol):

b. Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Each sample, standard, blank and optional control sample should be assayed in duplicate. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2°-8°C sealed tightly.

c. Wash the microwell strips twice with approximately 400 μl Wash Buffer per well with thorough aspiration of microwell contents between washes. Allow the Wash Buffer to sit in the wells for about 10 – 15 seconds before aspiration. Take care not to scratch the surface of the microwells. After the last wash step, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess Wash Buffer. Use the microwell strips immediately after washing. Alternatively microwell strips can be placed upside down on a wet absorbent paper for not longer than 15 minutes. Do not allow wells to dry.

d. Standard dilution on the microwell plate (Alternatively the standard dilution can be prepared in tubes - see 9.6.1): Add 100 μl of Sample Diluent in duplicate to all standard wells. Pipette 100 μl of prepared standard (see Preparation of Standard 9.6, concentration = 64 ng/ml) in duplicate into well A1 and A2 (see Table 1). Mix the contents of wells A1 and A2 by repeated aspiration and ejection (concentration of standard 1, S1 = 32 ng/ml), and transfer 100 μl to wells B1 and B2, respectively (see Figure 7). Take care not to scratch the inner surface of the microwells. Continue this procedure 5 times, creating two rows of human Bcl-2 standard dilutions ranging from 32.0 to 0.5 ng/ml. Discard 100 μl of the contents from the last microwells (G1, G2) used.

e)Add 100 μl of Sample Diluent in duplicate to the blank wells.

f. Add 80 μl of Sample Diluent to the sample wells.

g. Add 20 μl of each sample in duplicate to the sample wells.

h. Prepare Biotin-Conjugate (see Preparation of Biotin-Conjugate 9.4).

i. Add 50 μl of Biotin-Conjugate to all wells.

j. Cover with an adhesive film and incubate at room temperature (18 to 25°C) for 2 hours, if available on a microplate shaker set at 100 rpm.

k. Prepare Streptavidin-HRP (refer to Preparation of Streptavidin-HRP 9.5).

l. Remove adhesive film and empty wells. Wash microwell strips 3 times according to point c. of the test protocol. Proceed immediately to the next step.

m. Add 100 μl of diluted Streptavidin-HRP to all wells, including the blank wells.

n. Cover with an adhesive film and incubate at room temperature (18° to 25°C) for 1 hour, if available on a microplate shaker set at 100 rpm.

o. Remove adhesive film and empty wells. Wash microwell strips 3 times according to point c. of the test protocol. Proceed immediately to the next step.

p. Pipette 100 μl of TMB Substrate Solution to all wells.

q. Incubate the microwell strips at room temperature (18° to 25°C) for about 10 min. Avoid direct exposure to intense light. The colour development on the plate should be monitored and the substrate reaction stopped (see next point of this protocol) before positive wells are no longer properly recordable. Determination of the ideal time period for colour development has to be done individually for each assay. It is recommended to add the stop solution when the highest standard has developed a dark blue colour. Alternatively the colour development can be monitored by the ELISA reader at 620 nm. The 23 BMS244/3 and BMS244/3TEN human Bcl-2 substrate reaction should be stopped as soon as Standard 1 has reached an OD of 0.6 – 0.65.

r. Stop the enzyme reaction by quickly pipetting 100 μl of Stop Solution into each well. It is important that the Stop Solution is spread quickly and uniformly throughout the microwells to completely inactivate the enzyme. Results must be read immediately after the Stop Solution is added or within one hour if the microwell strips are stored at 2 - 8°C in the dark.

s. Read absorbance of each microwell on a spectro-photometer using 450 nm as the primary wave length (optionally 620 nm as the reference wave length; 610 nm to 650 nm is acceptable). Blank the plate reader according to the manufacturer's instructions by using the blank wells. Determine the absorbance of both the samples and the standards. Note: In case of incubation without shaking the obtained O.D. values may be lower than indicated below. Nevertheless the results are still valid