1. Michael A. Richardson, Laura E. Via, Daniel Schimel, Michelle Sutphin, Danielle Weiner, Kathleen England, Emmanuel Dayeo, Clifton E. Barry 3rd.
Tuberculosis Research Section, Laboratory of Clinical Infections Disease; Comparative Medicine Branch, National Institute of Allergy and Infectious
Disease, NIH, Bethesda Maryland, USA; Colorado State University School of Veterinary Medicine, Fort Collins, CO.
Experimental Design and Methods
Tuberculosis (TB) is a major health concern in developing nations with a high
population of low- and middle-income individuals. Compounding this socioeconomic
factor is an increasing prevalence of diabetes among the same population. The
weakened immune state of individuals with chronic hyperglycemia puts them at an
increased risk of developing an active TB infection. One of the major problems with
developing drug therapies to combat diabetes and TB co-infection is the difficulty of
developing a diabetic model that also expresses the pathologic hallmarks of human
TB infection. In this study we will attempt to combine radiologic – computed
tomography (CT) and 2-deoxy-2-[18F]-fluoro-D-glucose (FDG) Positron Emission
Tomography (PET) – methods developed in the common marmoset (Callithrix
janchus) to assess the effectiveness of streptomycin and isoniazid (SH) and
isoniazid, rifampicin, pyrazinamide and ethambutol (HRZE) cocktails in a novel
Type 1 and Type 2 diabetic New Zealand rabbit model. Alloxan will be used to
obtain pancreatic beta-cell ablation and induce insulin deficiency. To induce insulin
resistance, a custom high fat high carbohydrate diet (HFHC) based on the Harlan
2030 rabbit chow containing 35% fat, 44% carbohydrate, 21% protein (% k/cal) will
be fed to rabbits for 6 – 8 weeks prior to ALX adminsitration. We hope to use this
novel diabetic rabbit model as a foundation to asses not only the effectiveness of
SH and HRZE drug therapies, but to also determine and characterize the difference
in pathogenicity and latency between individuals infected with TB alone and those
with diabetes and TB co-infection.
Creating a diabetic rabbit model to assess radiologic responses to sterilizing
chemotherapy in diabetes and tuberculosis co-infection
Introduction
Conclusions
A
Induction of Type 1 Diabetes
To create an insulin deficient state, 8 New Zealand rabbits weighing in at 2.2 –
2.4kg were split into two groups and given an IV injection of 100mg/kg Alloxan
(ALX) via catheterized marginal ear vein. A 10% ALX suspension was created using
.9% injectable sodium chloride within 10 minutes of injection. Rabbits were
restrained in a standard cat restraining bag with eyes covered to reduce stress and
avoid the use of sedatives, allowing the ALX to be delivered over 2 – 3 minutes.
Using an AlphaTrak (Abbott Laboratories, USA) blood glucose meter calibrated for
humans, glucose levels were monitored prior to ALX injection and every 2 hours
thereafter, up to 6 hours. In order to avoid complications from the transient
hypoglycemic phase caused by ALX, food and water were available ad libitum prior
to injection, 5% sucrose was added to water bottles for 48 hours after ALX injection
and rabbits were given 6mL of 5% dextrose SQ.
Induction of Type 2 Diabetes
To induce insulin resistance, rabbits will be placed on a custom high fat high
carbohydrate (HFHC) diet for 6 – 8 weeks. This hybrid diet is based on the Harlan
2030 rabbit chow, and consists of 35% fat, 44% carbohydrate and 21% protein. The
fat portion contains 40% saturated fatty acids, 46% monounsaturated fatty acids,
and 14% polyunsaturated fatty acids from beef tallow and Primex vegetable
shortening. 18% of the carbohydrate portion is made up of 9% sucrose and 9%
fructose to simulate a typical western diet. Insulin resistance will be confirmed by
comparing pre- and post- oral glucose tolerance tests (OGTT), and supplemented
with data from daily non-fasting glucose monitoring. Once insulin resistance has
been confirmed, animals will be subjected to a 100mg/kg injection of ALX to destroy
beta-cell mass and mimic the pathogenicity of advanced type 2 diabetes.
Monitoring and Supporting Diabetic Rabbits
ALX causes a transient and sometimes severe hypoglycemia for up to 24 hours
after initial injection. The mechanism behind this transient hypoglycemia is unclear,
and careful monitoring is necessary to avoid hypoglycemic shock. Rabbits were
provided with food and water ad libitum prior to and after ALX injection, water
bottles were supplemented with 5% sucrose for 48 hours, and 5% dextrose was
administered SQ after ALX administration. In preparation of hypoglycemic shock,
Karo syrup, lactated ringers and syringes with 5% dextrose were readily available.
Glucose was monitored with the handheld AlphaTrak glucose monitor. Despite this
meter being calibrated for human use, it provided consistent glucose readings with
the rabbits. However, the meter has an upper limit of 600mg/dL and does not allow
for accurate readings above that. In future, this meter will have to be validated
against a chemistry analyzer, or a rabbit specific meter will have to be sourced.
Once hyperglycemic, rabbits were treated with Lantus (Insulin glargine; Sanofi-
Aventis, USA) once daily. Using a 50 unit insulin syringe, Lantus was injected SQ
into the scruff; a small portion of hair was shaved to ensure accurate injections.
Lantus dose was determined on a trial and error basis, as individual differences to
ALX and insulin inhibited the use of a standardized insulin dose per kilogram of
body weight.
Physiologic parameters tracked include: weight, non-fasting glucose, water intake,
food consumption and glucose tolerance. Animals off feed for any reason had their
diet supplemented with apples, carrots, alfalfa hay or Criticare (ClearH2O, USA).
Any rabbit determined to be dehydrated was given 50 – 100mL of LRS
subcutaneously. Animals that showed signs of pain were given a 1mg/kg SQ
injection of Ketoprophen (Zoetis, USA). Ketoprophen was given a maximum of
twice a week in order to avoid renal and gastrointestinal complications.
Tuberculosis Infection
Rabbits will be placed in an aerosol
chamber and exposed by
respiratory exposure to Mtb or M.
bovis. Virulent Mtb strains to be
utilized include H37Rv, CDC1551,
HN5 and HN878. Control animals
will be exposed to vehicle only –
tissue culture grade phosphate
buffered saline. Rabbits too large
to be placed in the nebulizer will be
given an intravenous injection of
1000 to 1,000,000 colony forming
units via marginal ear vein.
Therapeutic Models
Rabbits on study will be administered new drug compounds PO or with a
French feeding tube. Gavage will be avoided when possible. Compounds will
be mixed with raspberry syrup to offset the bitter taste, however, compounds
that are not willingly ingested will require the use of gavage under veterinary
supervision. Compounds may also be dosed in pill or capsule form, and will
contain the same amount of drug as the liquid PO doses. The pill or capsule
will be pharmaceutical grade and may be mixed with jelly to increase
palatability.
PET/CT Imaging
Rabbits will be lightly anesthetized and placed on an isofluorane mask for
anesthetic maintenance. Animals will receive a FDG PET/CT or CT alone to
classify their disease and render a 3D model. Optimal energy profile and soft
tissue resolution has been predetermined in order to obtain the most useful
image for lesion quantification.
Results
Diabetic Rabbit Model
• All 8 rabbits subjected to ALX became consistently hyperglycemic
within 48 hours
• Blood glucose levels were immediately elevated for up to 6 hours
after initial ALX administration
• Hypoglycemia did not occur until after 8 hours post injection –
outlining the need to ensure preventative measures are taken to
stave off hypoglycemic shock when caretakers are not present
• Of the 8 rabbits induced, 1 rabbit was found recumbent 24 hours
after ALX injection – this rabbit made a full recovery after treatment
with Karo syrup and a SQ dextrose injection
• Individual differences to insulin glargine therapy required constant
blood glucose monitoring after insulin administration – glucose
patterns for each rabbit must be understood
• Some animals required BID insulin dosing to control blood glucose
• All 8 rabbits showed signs of pollakiuria within 72 hours and
subjective signs of PU/PD within 96 hours
Non-fasting glucose for each animal in the Type 1 diabetic group. All
rabbits maintained a hyperglycemic state 48 hours after Alloxan injection.
Representative animals from each group demonstrate the long-term
effects of Lantus on hyperglycemic rabbits. Lantus was able to bring
glucose levels to baseline for greater than 6 hours.
0 1 2 3 4 5 6 7
0
100
200
300
400
500
600
700
Hours
BloodGlucose(mg/dL)
Single Dose Lantus
569
570
573
574
• Alloxan is a viable alternative to Streptozotocin for induction of
diabetes in rabbits
• A transient hypoglycemic phase occurs more than 8 hours post-
Alloxan injection
• Sucrose supplemented water and SQ dextrose are useful to combat
hypoglycemic shock during the hypoglycemic phase
• Hyperglycemic rabbits respond well to single-dose Lantus, providing
glycemic control for greater than 6 hours
Moving Forward
• Due to the amount of available time, not all aspects of the study were
completed at the time of writing
• The current group of 8 Type 1 diabetic rabbits will be infected with Mtb
after 4 weeks of hyperglycemia
• HRZE and SH drug cocktails will be compared, and subsequent
imaging taken for analysis
• Due to differences in blood glucose storage, it will be necessary to
either validate the human calibrated glucose meter or source a rabbit
specific glucose meter
C
Imaging developed in the common
marmoset can be translated to our
diabetic and tuberculosis co-infected
rabbits. These images allow for the
visualization of lesion burden and the
quantification of lesions, allowing for
the characterization of diabetic and
tuberculosis co-infected individuals
0 5 10 15
0
100
200
300
400
500
600
700
Day
BG(mg/dL)
Group 1 non-fasting Glucose
568
569
570
571
Group Avg
0 5 10 15
0
100
200
300
400
500
600
700
Day
BG(mg/dL)
Group 2 non-fasting Glucose
572
573
574
575
Group Avg
D2A D2P D3A D3P D6A D6P D10AD10PD13AD13P
0
200
400
600
800
BloodGlucose(mg/dL)
Glycemic Control
574
575
B